On graduation, she became an account manager selling soap powder for a pharmaceutical company. But while on holiday with friends, she decided this was no longer for her and she left to try something different.
In 2002, Medcalf landed the role of Sam Mitchell, taking over from Danniella Westbrook. She filmed her first scenes with Barbara Windsor in Spain. Just months after joining EastEnders, she was involved in a horrific car accident in France. After being cut-out of her Toyota Yaris, which was being driven by her then boyfriend, the accident left her with multiple broken bones, the worst being her leg, which had to be pinned together using a metal rod. Medcalf opted to leave Eastenders in 2005. Her final scenes were filmed in September 2005 and screened in November of that year. Her character, Sam, having escaped possible imprisonment for being an accessory in the murder of Den Watts, left to join her brother, Grant Mitchell, in Brazil.
Medcalf's first post-EastEnders work included a role in a dramatic staged reading of The Penelopiad directed by Phyllida Lloyd at St James's Church, Piccadilly on October 23, 2005, appearances for Children in Need on 18 November 2005 and in South Pacific at the Symphony Hall on 29 & 30 November 2005. In Spring 2006, she began an acclaimed four month run in the revival of Hay Fever at the Theatre Royal Haymarket alongside Judi Dench.Medcalf was the host of the Mitchells Weekend on the UKTV Gold network and on the 23 January 2007, she appeared in an episode of the BBC's Afternoon Play series called Death becomes him. In February and March 2007, Metcalf appeared as the "guest star" in a short UK tour of the West end hit The Play What I Wrote. On 2 April 2007, Medcalf took over the role of Sally Bowles from Anna Maxwell Martin in Rufus Norris' revival of Cabaret at the Lyric Theatre in the West End. Her run as Bowles ended on 29 September 2007.
From 25 February until the - 1 March 2008, Medcalf appeared at the New Victoria Theatre, Woking, in a run of The Vagina Monologues alongside Sue Holderness and Shobna Gulati. In March 2008 she appeared in an episode of The Fixer for ITV1, and then played a lead role in Harley Street, a daytime series for ITV1.
In March 2009, it was reported that EastEnders bosses were planning a return of Sam Mitchell, but they were trying to lure back Danniella Westbrook. It was confirmed by the BBC in April 2009, that Westbrook would be returning as Sam in the Summer of 2009, not Medcalf.