He is best known for his roles in the science fiction television series Doctor Who, where he played the third incarnation of the Doctor from 1970 to 1974, and as the title character in the series Worzel Gummidge. He also hosted the murder mystery quiz programme Whodunnit! between 1974 and 1978.

Pertwee was also a comic actor, with roles such as the conniving Chief Petty Officer Pertwee in The Navy Lark on BBC Radio and in another radio comedy series Waterlogged Spa. He also played the part of Lycus in the 1963 London stage production of A Funny Thing Happened on the Way to the Forum and appeared in the smaller role of Crassus in the 1966 film version. He appeared in four Carry On films: Carry On Cleo (1964), Carry On Screaming (1966), Carry On Cowboy (1965) and Carry On Columbus (1992). He also guest starred in the British comedy television series The Goodies in the episode "Wacky Wales".

On 14 April 1971, Pertwee was the subject of Thames Television's This Is Your Life.

In 1972, he released a vocal version of the Doctor Who theme music entitled "Who is the Doctor", and in 1980 he released a novel track based on Worzel Gummidge entitled "Worzel's Song".

Pertwee was an officer in the Royal Navy, spending some time working in naval intelligence during the Second World War. He was a crew member of HMS Hood and was transferred off the ship shortly before it was sunk, losing all but three men. He was married twice, first to Jean Marsh (1955-1960), whom he divorced, and then, on August 13, 1960, to Ingeborg Rhoesha, by whom he had two children, Sean and Dariel. He was a cousin of actor Bill Pertwee.

He returned to the role of the Doctor in the 1983 20th-Anniversary television movie The Five Doctors and in the 1993 charity special Dimensions in Time. During the 1990s, he made a guest appearance in the "Lords and Ladies" episode of the BBC Radio 4 comedy series Harry Hill's Fruit Corner, playing a Time Lord.

Pertwee would continue to act in films and television as well as make appearances world-wide in support of Doctor Who. Eventually, he became more aggressive in boosting projects that he favoured. Early success in persuading Doctor Who actors such as Patrick Troughton to appear as guests at American science fiction conventions inspired Pertwee to lobby for a radio version of the series after it was put on hiatus. Additionally, he vigorously canvassed British producers on behalf of Worzel Gummidge.

Ultimately, Pertwee was successful in seeing the Third Doctor return to the airwaves with two audio productions for BBC Radio, The Paradise of Death and The Ghosts of N-Space. Following its British cancellation, Worzel Gummidge was eventually picked up for production on New Zealand television in the late 1980s after much campaigning by Pertwee.

Pertwee continued on the convention circuit and with his voice and television acting until his death, aged 76, from a heart attack whilst on holiday in Connecticut on 20 May 1996 (some reports, however, place the location of his death in New York).

He died only days after the American broadcast of the Doctor Who television movie which used in its opening credits a logo based on the one from his era of the television series. The BBC broadcast of the television movie featured a dedication to Pertwee at its conclusion.

His last association with the series was posthumous. With the approval of his widow, Ingeborg, his voice was utilised as part of the plot of the Big Finish Productions 40th-Anniversary Doctor Who audio drama, Zagreus. Pertwee's voice was culled from a fan-produced Doctor Who film (see ]) Devious, portions of which were recorded prior to his death.