Rhona Mitra's first main role came as Scott Wolf's illicit love interest on Party of Five. In 2000, Mitra had a small, key role in the film Hollow Man, as the rape victim of Kevin Bacon, an incident that drives his character insane. She had a main role in medical drama Gideon's Crossing, as Dr. Alejandra 'Ollie' Klein. Rhona Mitra then had roles in Ali G Indahouse; Sweet Home Alabama; Stuck on You, and leading roles in Highwaymen and Spartacus. Rhona Mitra appeared in the final season of The Practice as Tara Wilson, and continued that role into its spin-off Boston Legal, but left not long into the second season. In 2005, Rhona Mitra played the role of Kit McGraw during Season 3 of Nip/Tuck. Rhona Mitra then went on to appear in Skinwalkers, The Number 23 and Shooter. In 2008, Rhona Mitra starred in the lead role of the science fiction/action film Doomsday as Major Eden Sinclair, and in 2009 went on to star in Underworld: Rise of the Lycans as Sonja, the daughter of the powerful vampire elder Viktor (played by Bill Nighy).
Rhona Mitra appeared as the live action model for Lara Croft, the lead character in Eidos Interactive's Tomb Raider video game series. She was ranked #46 on the Maxim Hot 100 Women of 2001.