Dushku was born in Watertown, Massachusetts, the daughter of Philip Richard George Dushku, an administrator and teacher in the Boston Public Schools, and Judith Ann "Judy" Dushku (née Rasmussen), a political science professor. Dushku's father is an Albanian from the city of Korçë and her mother is of Danish and English descent.
Dushku has three brothers, and was raised Mormon. She attended Beaver Country Day School in Chestnut Hill, Massachusetts, and graduated from Watertown High School.
Dushku came to the attention of casting agents when she was 10. She was chosen in a five-month search for the lead role of Alice in the film That Night. In 1993, Dushku landed a role as Pearl alongside Robert De Niro and Leonardo DiCaprio in This Boy's Life, a role that she said opened a lot of doors. The following year, she played the teenage daughter of Arnold Schwarzenegger and Jamie Lee Curtis in True Lies. She had parts as Paul Reiser's daughter in Bye Bye Love, as Cindy Johnson in Race the Sun, and roles in the television movie Journey and the short film Fishing with George.
Dushku intended to attend Suffolk University in Boston, where her mother taught at the time, but her agent asked her to submit a videotape audition for a show starring another of his clients, Sarah Michelle Gellar. After reading the script, Dushku rushed to a local Claire's to purchase dark makeup and other appropriate accessories for the part, Faith on Buffy the Vampire Slayer. When she began her work on that series, Dushku was still a minor, and had to receive emancipation to work the production's long hours. She later recalled with amusement that the judge who handled her emancipation case, who was an avid fan of that show, jokingly said that she would sign the emancipation order if she could get a signed photo from Dushku.
After completing high school, Dushku returned to acting with the role of Faith Lehane on Buffy the Vampire Slayer, a Slayer much more troubled than the main character Buffy Summers. Though initially planned as a five-episode role, the character became so popular that she stayed on for the whole third season and returned for a two-part appearance in season four, after which the remainder of her original story arc was played out as part of the first season of the Buffy spin-off series Angel. Repentant and rededicated, Faith returned as a heroine in other episodes of Angel and in the last five episodes of Buffy. Dushku was inundated with piles of fan mail from legions of prisoners. She said:
I've been getting fan mail from maximum security penitentiaries and death row. What are the authorities thinking of in playing a show with young teenage girls to Death Row inmates? They write everything – disgusting things that you don't even want to know about. And they send me pictures – "Oh, here's a picture of me before I was incarcerated!" – and there's some guy sat on the sofa with a bottle of beer and a moustache, and a big gut. It's so creepy. Way more creepy than Buffy.
In 2000, Dushku starred in the hit cheerleader comedy Bring It On. She followed that up with Soul Survivors, reuniting her with Race the Sun co-star Casey Affleck. In 2001, she appeared in The New Guy with DJ Qualls and City by the Sea with Robert De Niro and James Franco. The latter film garnered attention from a wider adult audience and several good reviews. The same year, Kevin Smith invited Dushku to be a part of Jay and Silent Bob Strike Back.
In 2003, Dushku starred in the horror film Wrong Turn and The Kiss, an independent comedy-drama. Starting that same year, she starred in a new Fox supernatural drama, Tru Calling, where she played the main character, medical student Tru Davies. After having a grant pulled out from under her, Tru is forced to take a job at a local morgue where she discovers her power to "re-live" the previous day over again if one of the deceased asks for her help to change what has happened. Dushku turned down a role in a spin-off of Buffy The Vampire Slayer which would have been about Faith. She has had many roles as a "bad girl" in movies and relishes the opportunities. In an interview with Maxim in May 2001, Dushku says of her roles, "It's easy to play a bad girl: You just do everything you've been told not to do, and you don't have to deal with the consequences, because it's only acting."
Dushku starred in an Off-Broadway production entitled Dog Sees God from December 2005, playing "Van's sister", a character paralleled with Lucy Van Pelt from the Peanuts comic strip on which the play production is based. She quit in February 2006 along with other members of the cast amidst rumors of abuse from the producer, which were latery-drama for Fox. This was the second Fox pilot in which she was cast, but not broadcast. She appeared in the Simple Plan music video, "I'm Just a Kid", as the band's love interest, as well as Nickelback's video for "Rockstar".
On October 1, 2005, she announced at Wizard World Boston that shooting had begun for Nobel Son in which she would star with Alan Rickman, Danny DeVito and Bill Pullman. The movie was released at the 2007 Tribeca Film Festival. Another project is On Broadway, an independent movie filmed in her native Boston. The movie received positive reviews, with a few of them highlighting Dushku's performance.
Dushku has had roles in five video games. She voiced the role of Yumi Sawamura in the English language version of Yakuza for the PlayStation 2, which was published and developed by Sega, and released in September 2006. Dushku also stars as Shaundi, one of the lead characters in Saints Row 2, which was developed by Volition, Inc. and published by THQ. It was released in North America on October 14, 2008, for the PlayStation 3 and Xbox 360. She was the voice talent for the role of Rubi Malone, the main character in the game WET. She appeared at Spike TV's 2008 Video Game Awards in December 2008. Danielle Nicolet took over the role of Shaundi in Saints Row: The Third.
Variety announced on August 2, 2006, that Dushku would co-star with Macaulay Culkin in Sex and Breakfast, a dark comedy written and directed by Miles Brandman. A reviewer described Dushku as "charming" and giving the character "an edge". The movie was released in Los Angeles on November 30, 2007, and on DVD on January 22, 2008. She starred in Open Graves, a 2008 horror-thriller about a satanic game co-starring Mike Vogel. She played the main character in The Thacker Case and The Alphabet Killer, both thrillers based on real-life events, one of them directed by Rob Schmidt, with whom she had worked on Wrong Turn. Both movies were released in 2008. The Alphabet Killer contains Dushku's first topless scene. The film earned mixed reviews, but reviewers praised Dushku's performance, commenting "Eliza Dushku commands the screen but cannot reconcile the script's conflicted and increasingly idiotic agendas." She appeared in Bottle Shock, a drama about Napa valley wine. The film was directed by Randall Miller, who helmed Nobel Son.
On August 26, 2007, Dushku signed a development deal with Fox Broadcasting Company and 20th Century Fox. Under the pact, the network and the studio would develop projects tailor-made for the actress as well as approach her with existing pitches and scripts.
Subsequently, it was announced on October 31 that Dushku had lured Joss Whedon, of Buffy the Vampire Slayer, back to TV, as they agreed to create a show called Dollhouse. Dushku produced the show and played the main character, 'Echo', which aired on Fox during the 2008–09 TV Season. In an interview, Dushku talked about how Dollhouse and her reconnection with Whedon came about:
I invited Joss Whedon to lunch after I did the business deal with Fox. We'd had a cool relationship in the past and I so wanted to do something else, and I wanted to get back into a television show. I had him on the brain for sure but I hadn't called him yet, but I sort of took a leap of faith and set things up with Fox and then called Joss. We went to a four-hour lunch where I just sort of used my womanly wiles. No, we've become such good friends, kind of like brother and sister and kind of like he was my watcher, my handler from when I first moved out to L.A. when I was 17 and I was a little bit of a wild child. He's watched me and helped me and taught me over the years. I told him how bad I wanted and needed him back and he accepted and here we are.
Dushku described Whedon as "my favorite genius ... favorite friend ... big brother ... and the only person out here I've ever wholeheartedly trusted, because he's never let me down." Dollhouse was renewed for a second season. The producers cited their confidence in the strength of Joss Whedon's fan base and high DVR numbers as their reasons for keeping the show. Fox cancelled Dollhouse on November 11, 2009. The show officially wrapped filming on the second and final season on December 16, 2009.
Dushku was the voice actor for contract killer "Rubi Malone" in the action video game Wet. She also secured exclusive rights to make The Perfect Moment, a film based on the life of photographer Robert Mapplethorpe and enlisted the help of Ondi Timoner.
In 2010, Dushku provided her voice for Noah's Ark: The New Beginning and appeared in the film Locked In. She also guest-starred in an episode of CBS' comedy The Big Bang Theory which aired on November 4, 2010.
In 2011, Dushku featured alongside Jayson Floyd in "One Shot", a short action clip on YouTube directed by and starring Freddie Wong, which was released on May 13, 2011. In August of the same year, Dushku visited Albania with a crew from the Travel Channel and Lonely Planet to film a documentary entitled Dear Albania, promoting tourism in her father's country of origin.
Dushku had a lead role in an online animated "motion comic" series, titled Torchwood: Web of Lies, based on the BBC series Torchwood: Miracle Day.
In June 2012, Dushku starred with Katie Cassidy, Gina Gershon and Michelle Trachtenberg in The Scribbler directed by John Suits and produced by Gabriel Cowan.
In 2013, she was cast as Patricia Holm in a pilot film for a proposed TV revival series of The Saint, but the series was not commissioned. In July 2017 the pilot was released on digital HD/VOD.
From 2013 to 2015, Dushku voiced the role of She-Hulk in the Disney XD animated series Hulk and the Agents of S.M.A.S.H..
In 2016, she had a recurring role in the fourth and final season of the Cinemax TV series Banshee.
In 2017, she had a recurring guest role in the last three episodes of the first season of the CBS drama series Bull with the option of becoming a series regular in season two. Additionally, her production company in association with IM Global Television was reported to be developing Glen Cook's The Black Company series of books for a television series with Dushku starring as The Lady.
Dushku was raised in the Mormon faith.
Dushku visited her father's family in Albania in 2006 after receiving an invitation from Albanian Prime Minister Sali Berisha. She visited Kosovo and received an Albanian Eagle tattoo on the back of her neck. While on her second visit to Albania in 2011, she applied for Albanian citizenship and obtained the Albanian passport and ID Card. She became an honorary citizen of Tirana, and was given the honorary title of Tirana Ambassador of Culture and Tourism in the World by Tirana mayor Lulzim Basha. Additionally, she was given honorary citizen status in her father's home town of Korçë, Albania.
She is the founder and CEO of Boston Diva Productions and serves on the board of directors of the THRIVEGulu organization (The Trauma Healing and Reflection Center in Gulu), an organization dedicated to helping the survivors of war (including former child soldiers) in Northern Uganda. As a role model to campus leaders for her activism, Dushku was invited by the Millennium Campus Network (MCN) as a national keynote speaker and honored as a Global Generation Award winner alongside U.S. Secretary of State John Kerry at MCN events in 2011.
Dushku has stated that she has attention deficit hyperactivity disorder (ADHD).
She began dating former Los Angeles Lakers basketball player Rick Fox in October 2009, and in August 2010 the couple confirmed that they were living together. It was announced on June 24, 2014 that the pair had split.
Dushku announced in December 2011, that she had decided to switch to a vegan diet after watching the documentary Forks over Knives. In December 2014 she said on her Twitter page she was no longer a vegetarian.
Dushku, who had originally intended to attend college in Boston before her big break on Buffy altered those plans, announced in March 2014 at the Emerald City Comicon that she would be attending college later that year.
Dushku previously resided in Laurel Canyon, Los Angeles, California. On June 22, 2014, The Boston Globe reported that Dushku had left Los Angeles, moved back to Boston, and was planning to attend college. She is currently a student at Suffolk University, studying sociology.
Dushku is politically active and campaigned for Bernie Sanders in the U.S. presidential election of 2016.
In June of 2017, she became engaged to Peter Palandjian.
Her performance in Tru Calling garnered her two nominations in 2004: for a Teen Choice Award as Choice Breakout Star – Female, and for a Saturn Award as Best Actress in a Television Series. Maxim magazine ranked Dushku 6th on the "Hot 100 Women of 2009" list. Dushku was nominated in 2009 for a Scream Award for Best Science Fiction Actress for her role of Echo, and she was named Ambassador for the 2012 Consumer Electronics Show Entertainment Matters program in October 2011.