Buono was born in San Diego, California, the son of Myrtle Belle (née Keller; 1909–1979) and Victor Francis Buono (1907–1981). His maternal grandmother, Myrtle Glied (1886–1969), was a Vaudeville performer on the Orpheum Circuit. When he was a boy, she taught him songs and recitations and encouraged him to perform for visitors. Even though the young Buono enjoyed the polite applause of those captive audiences, he aspired to be a doctor. When he was sixteen, Father John Aherne, OSA, of St. Augustine High School in San Diego cast him as Papa Barrett in the play The Barretts of Wimpole Street. Buono appeared in three plays a year during high school, including Aladdin and His Wonderful Lamp and Shakespearean dramas such as the play Hamlet. Buono played the role of King Claudius.
He started appearing on local radio and television stations, and at age 18 joined the Globe Theater Players in San Diego. The director had confidence in Buono and cast him in Volpone, A Midsummer Night's Dream and other Globe presentations. He received good notices for his various Shakespearean roles and in modern plays such as The Man Who Came to Dinner and Witness for the Prosecution.
In the summer of 1959, a talent scout from Warner Bros. saw the heavy-set Buono play Falstaff at the Globe and took him to Hollywood for a screen test. Buono made his first network TV appearance playing the bearded poet Bongo Benny in an episode of 77 Sunset Strip. Over the next few years, he played menacing heavies in nearly every Grade "A" private eye series on TV and also appearing on The Untouchables. After appearing in a few uncredited film roles, he was cast by director Robert Aldrich in the psychological horror movie What Ever Happened to Baby Jane? (1962). The film starred Bette Davis and Joan Crawford, and Buono played the ne'er-do-well musical accompanist Edwin Flagg, a performance for which he was nominated for the Academy Award for Best Supporting Actor and the Golden Globe Award for Best Supporting Actor – Motion Picture.
Shortly after What Ever Happened to Baby Jane?, Buono appeared in Hush… Hush, Sweet Charlotte (1964) as Big Sam Hollis, the father of Bette Davis, who played the title role. The film was also directed by Aldrich. In the Biblical epic The Greatest Story Ever Told (1965), Buono portrayed the High Priest Sorak, and in The Strangler, a film based on the actual Boston Strangler Murders of the time, he portrayed Leo Kroll.
He also appeared in 4 for Texas (1963), Robin and the 7 Hoods (1964), The Silencers (1966), Who's Minding the Mint? (1967), Target: Harry (1969), Beneath the Planet of the Apes (1970), The Mad Butcher (1972) and The Evil (1978).
Though Buono had a vast body of work in movies, he also had extensive television appearances to his credit; one was in the recurring role of Count Manzeppi in CBS's The Wild Wild West. He also played unrelated characters in that series' premiere episode and in the second and final Wild Wild West reunion movie, More Wild Wild West (1980).
Buono was cast to play villains of various ethnic origins on many television programs between 1960 and 1970. He was cast twice in 1960 in the ABC western series, The Rebel, starring Nick Adams, in the episodes "Blind Marriage" and "The Earl of Durango." In 1962, he played Melanthos Moon in an episode of ABC's The Untouchables, titled "Mr. Moon", where he played a San Francisco art and antique dealer who hijacked a supply of the paper used for printing United States currency. In a 1963 episode of the same series, titled The Gang War, he played Pamise Surigao, a liquor smuggler competing with the Chicago mob.
In the episode "Firebug" (January 27, 1963) of the CBS anthology series GE True, hosted by Jack Webb, Buono plays a barber in Los Angeles, who is by night a pyromaniac. In the storyline, the United States Forest Service believes one arsonist is causing a series of fires in California. The episode also starred Keith Andes and Arch Johnson.
Buono appeared in four episodes of CBS's legal drama Perry Mason. In season 5, 1962, he portrayed Alexander Glovatsky, a small-town sculptor, in "The Case of the Absent Artist". In season 7, 1964, he played murderer John (Jack) Sylvester Fossette in the episode "The Case of the Simple Simon". In season 8, 1965 he played murderer Nathon Fallon in "The Case of the Grinning Gorilla". In season 9, 1966, he appeared in the only color episode, "The Case of the Twice Told Twist", as Ben Huggins, the ringleader of a car-stripping ring.
Buono played the villain King Tut on the television series Batman. A Jekyll-and-Hyde character, William McElroy is a timid Yale professor of Egyptology who, after being hit in the head with a brick at a peace rally, assumes the persona of the charismatic, monomaniacal Egyptian King Tut. When he suffers another blow to the head, the villain recovers his meek academic personality. The role, which proved to be the most frequently featured original villain in the series, was one of Buono's favorites because he was delighted at being able to overact without restraint.
He played another villain in a 1967 unsold TV pilot film based on the Dick Tracy comic strip.
Buono also played a scientist bent on world domination in the Voyage to the Bottom of the Sea episode "The Cyborg"
Buono made a guest appearance as Hannibal Day in the Get Smart episode "Moonlighting Becomes You", originally airing January 2, 1970, and appeared three times as Dr. Blaine in the ABC sitcom Harrigan and Son, starring Pat O'Brien and Roger Perry as a father-and-son team of lawyers. He appeared in a segment of NBC's Night Gallery titled "Satisfaction Guaranteed". He also appeared in a 1973 episode of Hawaii Five-O (episode 15). He made two memorable appearances on ABC's The Odd Couple, once in the episode "The Exorcists" and again in "The Rent Strike", where he portrayed Mr. Lovelace. In 1976, he appeared in the NBC situation comedy The Practice, portraying Bernard on the episode "Jules and the Bum". He also made nine appearances on the 1977 NBC series Man from Atlantis, which starred Patrick Duffy as the series' title character of Mark Harris, appearing all nine times as Mr. Schubert, Mark's greatest enemy.
In the 1970s, Buono released several comedy record albums which poked fun at his large stature, The first of which was Heavy!, as well as a book of comic poetry called It Could Be Verse. He began to style himself as "the fat man from Batman." During guest appearances on The Tonight Show Starring Johnny Carson, he frequently recited his poetry. The most popular of his poems was Fat Man's Prayer, a work often erroneously attributed to Dom DeLuise or Jackie Gleason. It included many widely quoted couplets such as:
We are what we eat, said a wise old man,
And Lord, if that's true, I'm a garbage can!
At oleomargarine I'll never mutter,
For the road to hell is spread with butter.
And cake is cursed, and cream is awful,
And Satan is hiding in every waffle.
Give me this day my daily slice—
In the late 1970s and in 1980, Buono played the millionaire father of the memory-impaired Reverend Jim Ignatowski on Taxi. Buono died before the end of the series and another actor briefly assumed the role. The character was eventually killed off, followed by an episode where Jim learns to cope with his father's death.
In 1980, Buono appeared in the television movie Murder Can Hurt You as Chief Ironbottom, a parody of the title character from Ironside. His later roles were more of pompous intellectuals and shady con men, although he also played straight roles. In the miniseries Backstairs at the White House (1979), he portrayed President William Howard Taft.
Buono was found dead at his home in Apple Valley, California, on New Year's Day 1982; he died of a sudden heart attack. He is entombed with his mother Myrtle in Greenwood Memorial Park in San Diego, but his name is not inscribed on the crypt.
Buono liked to read and write, and one of his main interests was Shakespeare. "The more you study him," he said, "the greater he grows." He was also highly regarded as a gourmet chef.
In regard to relationships (and the implicit questioning of his sexuality), Buono is quoted as saying, "I've heard or read about actors being asked the immortal question, 'Why have you never married?' They answer with the immortal excuse, 'I just haven't found the right girl.' Because I'm on the hefty side, no one's asked me yet. If they do, that's the answer I'll give. After all, if it was good enough for Monty Clift or Sal Mineo..." Buono was unusual among gay performers of his era by openly living together with same-sex partners, although he was not flamboyant about his lifestyle and referred to himself as a "conscientious objector" in the "morality revolution" of the 1960s.
Despite his weight, Victor Buono was known to be a playboy according to the commentary on the DVD edition of Hush... Hush, Sweet Charlotte.