In the early 1980s, Pertwee was cast as Captain Fitzpatrick in the play Tom Jones, based on the novel by Henry Fielding. After leaving Sunbury College, Pertwee trained at Bristol Old Vic Theatre School and, after graduating in 1986, toured with the Royal Shakespeare Company for three years. In 1999 he portrayed Brutus in the Hallmark Channel film Cleopatra. He co-owned the Natural Nylon film production company along with Sadie Frost, Jude Law, Jonny Lee Miller, and Ewan McGregor. The company folded in 2003. Pertwee is perhaps best known for his varying portrayals of grimacing death in many of his film roles, a skill that led to him being awarded 'best death face' at Bristol Old Vic.

Pertwee is the brother of Actress Dariel Pertwee, son of actor Jon Pertwee (of Doctor Who fame), grandson of actor and Screenwriter Roland Pertwee, and is more distantly related to actor Bill Pertwee. He married make-up Artist Jacqueline Jane ″Jacqui″ Hamilton-Smith, daughter of Anthony Hamilton-Smith on 12 June 1999 at the House of Lords. His wife gave birth to twins, Alfred and Gilbert, around Christmas 2001. They were born prematurely; Gilbert died four days later.

Pertwee has been a supporter of the Labour Party and did a party political broadcast in the run up to the 2010 UK general election. In the broadcast he makes reference to his father's resolve.

Pertwee has been in several dramatic works for BBC Radio 4, including playing the actor Oliver Reed in the play Burning Both Ends by Matthew Broughton in 2011.

In 2014, Pertwee was cast in Fox's TV series Gotham, a series presenting an origin for the characters of the Batman franchise. Pertwee portrays Alfred Pennyworth, a tough-as-nails ex-special forces from east London who works as the Wayne family's loyal butler. After Thomas and Martha Wayne are murdered, he becomes guardian and mentor to their young son Bruce – the boy who will one day become Batman.