Chambers was born in Doncaster, West Riding of Yorkshire, where she went to school at St. Mary’s. Her secondary education was at St Swithun’s School, Winchester, Hampshire. She then trained at the Webber Douglas Academy of Dramatic Art in the 1980s, where she was a classmate of actor Ross Kemp. Her sister is Sarah Doukas of Storm Model Management
After taking some smaller parts on television productions such as The Bill, in November 1994 Chambers played the role of Charity Pecksniff in the TV serialisation of the Charles Dickens novel Martin Chuzzlewit. In the same month she first played the role of Alice Tinker in the BBC comedy The Vicar of Dibley. Chambers appeared in all 24 episodes. In 1998 Chambers won the British Comedy Award for Best Actress for her performance. Her last outing as Alice was the Comic Relief Special in March 2007.
Chambers worked as a voice performer in the animated made-for-TV film The Wind in the Willows in 1995. In 2003 she provided the voice of Spotty for two episodes in the TV seriesLittle Robots. Her episodes were called “Spotty Rules” and “Spotty’s Clean Machine”.
She was in theatre for about 10 years before her major break in television. She has appeared in a number of stage productions including Tartuffe and Invisible Friends. In 2002 she toured with the Michael Frayn play, Benefactors, where she starred opposite Neil Pearson.