Mortimer was born in Hammersmith, London, England, to Sir John Mortimer, QC (a lawyer and writer who created the television series Rumpole of the Bailey), and his second wife, Penelope (née Gollop). She has a younger sister, Rosie; two older half-siblings, Sally Silverman and Jeremy, by her father's first marriage, to author Penelope Fletcher; and a half-brother, Ross Bentley, by her father's extramarital relationship with actress Wendy Craig.
Mortimer studied at St Paul's Girls' School in west London, where she appeared in several pupil productions. She then went on to Oxford University, where she read Russian at Lincoln College and performed in several plays. Before becoming an actress, Mortimer wrote a column for the Daily Telegraph, and was screenwriter for an adaptation of Lorna Sage's memoir, Bad Blood.
Mortimer performed for several plays while studying at Oxford University, and while acting in a student production she was spotted by a producer who later cast her in the lead in a television adaptation of Dame Catherine Cookson's The Glass Virgin (1995). Subsequent television roles included Sharpe's Sword and Coming Home. She followed this with the 1996 television film, Lord of Misrule, directed by Guy Jenkin and filmed in Fowey, Cornwall.
Her first feature film role was opposite Val Kilmer in The Ghost and the Darkness, also in 1996. She appeared in the Irish coming-of-age story The Last of the High Kings, also released in 1996.
In 1998 she appeared as Kat Ashley in Elizabeth, and played Miss Flynn in the television mini series Cider with Rosie, which was adapted for television by her father. In 1999, she played three roles that raised her profile outside the United Kingdom: She was the "Perfect Girl" dropped by Hugh Grant in Notting Hill; Esther in the television mini series Noah's Ark; and Angelina, the star of the film within a film in Scream 3.
In 2000, Mortimer was cast as Katherine in Kenneth Branagh's musical adaptation of Love's Labour's Lost, where she met actor and future husband Alessandro Nivola. She took on her biggest role in an American film to date, playing opposite Bruce Willis in Disney's The Kid. In 2002, she had a major role in The 51st State (also known as Formula 51), starring opposite Samuel L. Jackson and Robert Carlyle.
In 2003, Mortimer appeared in Stephen Fry's film Bright Young Things. In 2004, Mortimer played the female lead in the film Dear Frankie. In 2005, she played a major role as the oblivious spouse of Jonathan Rhys Meyers's adulterer in Woody Allen's Match Point, as well as voiced young Sophie in the English-dubbed version of Howl's Moving Castle. In 2007 she played a supporting role in Lars and the Real Girl as the supportive sister-in-law of Ryan Gosling's title character. She appeared in The Pink Panther in 2006 and in its 2009 sequel. In the last three episodes of the first season of 30 Rock, she played Phoebe, the mysterious love interest of Alec Baldwin's character, Jack Donaghy.
Mortimer played an aspiring actress opposite Andy García in City Island (2009) and as Rachel Solando in Martin Scorsese's 2010 film Shutter Island. She played Leonie Gilmour in Leonie, released in the autumn of 2010. In 2011, she had a role in Our Idiot Brother as Liz, the sister of Paul Rudd's character. In 2011, she also started work with the acclaimed screenwriter and producer Aaron Sorkin as Mackenzie McHale in HBO's The Newsroom. In January 2013 it was announced that Mortimer would be co-creating and starring in the comedy series Doll & Em for Sky Living, along with her longtime friend, actress and comedian Dolly Wells.
In 2000, Mortimer met American actor Alessandro Nivola, while both were starring in Love's Labour's Lost. The couple married in the village of Turville in the Chilterns, Buckinghamshire, on 3 January 2003. A Mexican punk band performed at their wedding. They have two children.