Berkoff was born Leslie Steven Berks on 3 August 1937, in Stepney in the East End of London. He is the son of Pauline (née Hyman), a housewife, and Alfred Berks (né Berkoff), a tailor. His family was Jewish (originally from Romania and Russia). Berkoff's father had anglicised his family surname to "Berks" in order to aid the family's assimilation into Britain. Berkoff later removed the “s” from and added back the "off" to his own name, and went by his middle name.

Berkoff attended Raine's Foundation Grammar School (1948–50), Hackney Downs School, the Webber Douglas Academy of Dramatic Art (1958) and L'École Internationale de Théâtre Jacques Lecoq (1965).

Berkoff started his theatre training in the Repertory Company at Her Majesty's Theatre in Barrow-in-Furness, for approximately two months, in June and July 1962.

As well as an actor, Berkoff is a noted Playwright and theatre Director, with a unique style of writing and performance. His earliest plays are adaptations of works by Franz Kafka: The Metamorphosis (1969); In the Penal Colony (1969) and The Trial (1971). In the 1970s and 1980s, he wrote a series of verse plays including East (1975), Greek (1980) and Decadence (1981), followed by West (1983) later adapted and recorded at Limehouse Studios for transmission on C4 television in 1983, Harry's Christmas (Lunch) also recorded at Limehouse Studios in 1983 was never transmitted by C4 as it was considered "too dark", Sink the Belgrano! (1986), Massage (1997) and The Secret Love Life of Ophelia (2001). Berkoff described Sink the Belgrano! as "even by my modest standards ... one of the best things I have done".

In television, Berkoff had early roles in episodes of The Avengers and UFO episode "The Cat with Ten Lives" in 1970. Other TV credits include: Hagath, in the episode "Business as Usual" of Star Trek: Deep Space Nine; Stilgar, in the mini-series Children of Dune; gangster Mr Wiltshire in one episode of Hotel Babylon; Lawyer Freddie Eccles in "By the Pricking of My Thumbs", an episode of Agatha Christie's Marple; and Adolf Hitler in the mini-series War and Remembrance. In 1998, he made a guest appearance in the Canadian TV series La Femme Nikita (in the episode "In Between").

In the Stanley Kubrick films A Clockwork Orange (1971) and Barry Lyndon (1975), Berkoff played a police officer, and a gambler aristocrat. His other films include the Hammer film Prehistoric Women (1967), Nicholas and Alexandra (1971), The Passenger (1975), Joseph Andrews (1977), McVicar (1980), Outland (1981), Coming Out of the Ice (1982), Underworld (1985), Revolution (1985), Absolute Beginners (1986), Prince's film Under the Cherry Moon (1986), Prisoner of Rio (1988), Fair Game (1995), the Australian film Flynn (1996), and Legionnaire (1998).

In film, Berkoff has played villains such as Soviet General Orlov in the James Bond film Octopussy (1983), the corrupt art dealer Victor Maitland in Beverly Hills Cop (1984), the Soviet officer Colonel Podovsky in Rambo: First Blood Part II (1985), and gangster George Cornell in The Krays (1990). Berkoff has stated that he accepts roles in Hollywood only to subsidise his theatre work, and that he regards many of the films in which he has appeared as lacking artistic merit.

In 1988, Berkoff directed an interpretation of Salome by Oscar Wilde, performed in slow motion, at the Gate Theatre, Dublin. For his first directorial job at the UK's Royal National Theatre, Berkoff revived the play with a new cast at the Lyttelton Auditorium; it opened in November 1989. In 1998, his solo play Shakespeare's Villains premièred at London's Haymarket Theatre and was nominated for a Society of London Theatre Laurence Olivier Award for Best Entertainment.

In the 1989 romantic comedy The Tall Guy, struggling actor Dexter King (Jeff Goldblum) auditions unsuccessfully for an imaginary "Berkoff play" called England, My England. In the audition, characters dressed as skinheads swear repetitively at each other and a folding table is kicked over. Afterwards, Dexter's agent Mary (Anna Massey) muses, "I think he's probably mad ..."

In 1991, Berkoff's play Kvetch won the Evening Standard Theatre Award for Best Comedy. In 1997, Berkoff won the first Total Theatre Lifetime Achievement Award. In 1998, he was nominated for a The Society of London Theatre's Laurence Olivier Award for Best Entertainment for his one-man show Shakespeare's Villains. In 1999, the 25th-anniversary revival of the play East, directed by Berkoff, received the Stage Award for Best Ensemble work at the Edinburgh Festival Fringe. In 2000, he won the LA Weekly Theater Award for Solo Performance, again for Shakespeare's Villains. Also in 2000, his play Messiah, Scenes from a Crucifixion received a Scotsman Fringe First Award at the Edinburgh Fringe Festival. In 2001, The Secret Love Life of Ophelia won a Bank of Scotland Herald Angel.

"I'm scared of Steven Berkoff" is a line in the lyrics of the song "I'm Scared" by Queen Guitarist Brian May, issued on his 1993 debut solo album Back to the Light. May has declared himself to be an admirer of Berkoff and his wife, Anita Dobson, has appeared in several of Berkoff's plays.

In 1994, he both appeared in and directed the film version of his verse play Decadence. Filmed in Luxembourg, it co-stars Joan Collins.

In 1996, Berkoff won Berkoff vs. Burchill, a libel civil action that he brought against Sunday Times Journalist Julie Burchill after she published comments suggesting that he was "hideously ugly". The judge ruled for Berkoff, finding that Burchill's actions "held him to ridicule and contempt."

Berkoff was the main character voice in Expelling The Demon (1999), a short animation with music by Nick Cave. It received the award for Best Film at the Ukraine Film Festival. He has a cameo in the 2008 film The Cottage. Berkoff appeared in the 2010 British gangster film The Big I Am as "The MC", and in the same year portrayed the antagonist in The Tourist. Berkoff portrayed Dirch Frode, attorney to Henrik Vanger (Christopher Plummer), in David Fincher's 2011 adaptation of The Girl with the Dragon Tattoo. Another 2011 credit is the independent film, Moving Target. He also stars in Katherine of Alexandria (2014) playing the role of Liberius.

According to Annette Pankratz, in her 2005 Modern Drama review of Steven Berkoff and the Theatre of Self-Performance by Robert Cross: "Steven Berkoff is one of the major minor contemporary dramatists in Britain and – due to his self-fashioning as a bad boy of British theatre and the ensuing attention of the media – a phenomenon in his own right." Pankratz further asserts that Cross "focuses on Berkoff's theatre of self-performance: that is, the intersections between Berkoff, the public phenomenon and Berkoff, the Artist."

In a Daily Telegraph travel article written while visiting Israel in 2007, Berkoff described Melanie Phillips' book Londonistan: How Britain is Creating a Terror State Within, as "quite overwhelming in its research and Common sense. It grips me throughout the journey."

Berkoff appeared in the British Heart Foundation's two-minute public Service advertisement, Watch Your Own Heart Attack, broadcast on ITV in August 2008. He also presented the BBC Horizon episodes, "Infinity and Beyond" (2010) and "The power of the Placebo" (2014).

Berkoff has spoken and written about how he believes Jews and Israel to be regarded in Britain. In a January 2009 interview with The Jewish Chronicle, in which he discussed anti-Israel sentiment in the aftermath of the Gaza War, he said:

Speaking to The Jewish Chronicle in May 2010, Berkoff criticised the Bible but added, "it inspires the Jews to produce Samsons and heroes and to have pride". Berkoff went on to say of the Talmud in the same article: "As Jews, we are so incredibly lucky to have the Talmud, to have a way of re-interpreting the Torah. So we no longer cut off hands, and slay animals, and stone women."

In 2012, Berkoff, with others, wrote in support of Israel's national theatre, Habima, performing in London.

In 2014 Berkoff played a supporting role in the second season of the Lifetime TV show Witches of East End.

In 2016, he appeared in Season 3, Episode 1 of Man Down on Channel Four in the UK.