Mary Barbara Jefford was born in Plymstock, Devon, the daughter of Elizabeth Mary Ellen (née Laity) and Percival Francis Jefford. She was brought up in the West Country and attended Weirfield School in Taunton, Somerset. She attended the Hartly-Hodder School of Speech Training and Dramatic Art before training at the Royal Academy of Dramatic Art in London, where she was awarded the Bancroft Gold Medal. In 1946, whilst still a student, she obtained small parts in the radio production of Westward Ho! and other radio plays, but her stage debut came in 1949, when she played the part of Viola in Twelfth Night at the Dolphin Theatre, Brighton.

Over the next four years she went on to play many more major Shakespearean roles: Anne Boleyn in Henry VIII in 1950; Calphurnia in Julius Caesar opposite Anthony Quayle and Michael Langham in 1950; Hero, opposite John Gielgud and Peggy Ashcroft in 1950; Lady Percy in Henry IV, opposite John Kidd, Anthony Quayle and Michael Redgrave in 1951; Isabel opposite Richard Burton in Henry V, in 1951; Desdemona to Anthony Quayle's Othello in 1952; Rosalind in As You Like It (New Zealand Tour, 1953); Lady Percy in Henry IV, Part 1 ( New Zealand Tour and International Tour, 1953); Hippolyta in A Midsummer Night's Dream in 1954; Kate to Keith Michell's Petruchio in The Taming of the Shrew in 1954; and Helen in Troilus and Cressida in 1954.

In 1959 she appeared as Ophelia in a TV production of Hamlet. For the James Bond film From Russia with Love (1963) she provided the uncredited voice of Tatiana Romanova, played by Daniela Bianchi. Jefford provided additional voice work in later Bond films, dubbing Molly Peters in Thunderball (1965) and Caroline Munro in The Spy Who Loved Me (1977). Her first major film role was as Molly Bloom in Ulysses (1967), for which she was nominated for a British Academy Award. This was followed by A Midsummer Night's Dream (1968), The Bofors Gun (1968), The Shoes of the Fisherman (1968) and Lust for a Vampire (1971) She played Magda Goebbels in Hitler: The Last Ten Days (1973). Other films include Nelly's Version (1983), Fellini's And the Ship Sails On (E la nave va) (1983), Claudia (1985), When the Whales Came (1989), The Saint, Roman Polanski's The Ninth Gate (1999) and Terence Davies's The Deep Blue Sea. In 2013, she played Sister Hildegard, a small but crucial part, in Stephen Frears's Philomena with Judi Dench and Steve Coogan.

In 1965, Jefford was awarded the OBE for her Service to the theatre, becoming the youngest ever civilian recipient of the award to that date.

Jefford next started a period of work with Frank Hauser's Oxford Playhouse which included the first of her three Cleopatras, Racine's Phèdre and Lina in Misalliance which transferred to the Criterion Theatre. In the early 1970s she played Katherine Stockman in "An Enemy of the People " at the Chichester Theatre Festival.<Program from production>

Jefford has appeared in several television dramas in the Play For Today series (Edna, the Inebriate Woman, 1971); and has appeared in several other series. These include Journey to the Unknown, which also aired in the US, in 1968; Walter and June (1986); Porterhouse Blue (1987); Mrs Herriton in Where Angels Fear to Tread (Charles Sturridge, 1991); The House of Eliott (1991); Midsomer Murders (2000) and Madame Bovary (2000). She has also appeared in episodes of The Ruth Rendell Mysteries, Campion and the Inspector Alleyn Mysteries. She also appeared in The Creeper a 2010 episode of Midsomer Murders.

Other West End plays included Ride A Cock Horse, Filumena, Mistress of Novices and The Dark Horse, as well as the Almeida Theatre's Racine Season at the Albery Theatre. With this company she also played her second Volumnia in Coriolanus, opposite Ralph Fiennes in London, New York City and Tokyo, her first being at Stratford with Charles Dance. In 1976 she was in the opening production at the Olivier Theatre playing Zabina in Tamburlaine the Great with Albert Finney.

In 1977 she was also awarded the Jubilee Festival Medal.

She has repeated many Shakespeare roles in her long career, appearing in 54 productions of all but four of his plays. The last of these was Michael Grandage's Richard III with Kenneth Branagh in 2002, at the Crucible Theatre, Sheffield in which she played Queen Margaret, opposite Derek Jacobi for the second time.

In July 2007 she played Mrs Higgins (the mother of Henry Higgins) in Peter Hall's acclaimed Theatre Royal, Bath production of George Bernard Shaw's Pygmalion, which transferred to the Old Vic in May 2008

After leaving Stratford she co-starred with Michael Redgrave, in Tiger at the Gates in the West End and on Broadway, before returning to work at the Old Vic. Amongst other roles she played there were Portia in The Merchant of Venice; Imogen in Cymbeline; Beatrice in Much Ado About Nothing; Julia in The Two Gentlemen of Verona; Tamora in Titus Andronicus; Lady Anne in Richard III; Viola in Twelfth Night; Queen Margaret in Henry VI 1–3 ; Isabella in Measure for Measure; Regan in King Lear; Rosalind in As You Like It; and Viola in Twelfth Night. In 1978 she played Gertrude to Derek Jacobi's Hamlet.