Mitra began her career as a model. She came to prominence as the Lara Croft model between 1997 and 1998. After completing her stint as Lara Croft, she concentrated on acting and is known for her roles as Holly Marie Begins on the sixth season of Party of Five (1999–2000); as Tara Wilson on the final season of The Practice (2003–2004) and the first and second seasons of Boston Legal (2004–2005); as Detective Kit McGraw on the third season of Nip/Tuck (2005); in the lead role of the science fiction/action film Doomsday as Major Eden Sinclair (2008); as Sonja in Underworld: Rise of the Lycans (2009); and as Major Rachel Dalton on the third and fourth seasons of Strike Back (2012–2013); and as Dr. Rachel Scott in the first two seasons of The Last Ship (2014–2015).

Mitra appeared as the live action model for Lara Croft, the lead character in Eidos Interactive's Tomb Raider video game series before Angelina Jolie took the role for the two Tomb Raider films (2001–2003). Mitra was ranked No. 46 on the Maxim Hot 100 Women of 2001. She played the romantic interest of Christopher Lambert in Beowulf. Her first main role came as Scott Wolf's illicit love interest on Party of Five. In 2000, Mitra had a small role in the film Hollow Man, as a neighbour sexually assaulted by Kevin Bacon's character. She had a main role in the medical drama Gideon's Crossing, as Dr. Alejandra "Ollie" Klein. Mitra then had roles in Ali G Indahouse, Sweet Home Alabama, Stuck on You, and leading roles in Highwaymen and Spartacus. 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, Mitra played the role of Kit McGraw during Season 3 of Nip/Tuck. Mitra then went on to appear in Skinwalkers, The Number 23 and Shooter.

Rhona Mitra was nominated for a Screen Actors Guild Award in 2006 from Boston Legal TV Series. She was also nominated in 2009 for Scream Awards for Best Actress from Underworld: Rise of the Lycans.

In 2008, Mitra starred in the lead role of the science fiction/action film Doomsday as Major Eden Sinclair (sometimes mistakenly named in some databases as being Caryn Peterson), 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). While filming, she grew fond of her vampire fangs, even declining to remove them when they couldn't be seen. "I put those fangs on the first day and I felt they should always have been there; it's strange. So I kept them in through the entire time of shooting, throughout all my dialogue and everything." She also appeared in three episodes of Stargate Universe.

She stars in the 2010 Anders Anderson thriller film Stolen, alongside Josh Lucas, Jon Hamm and James Van Der Beek. She portrayed Claire Radcliff in the 2010 ABC supernatural series The Gates. She played Major Rachel Dalton on Cinemax's series Strike Back: Project Vengeance, replacing Amanda Mealing.

In 2014 and 2015, she played Dr. Rachel Scott in the first two seasons of Michael Bay's post-apocalyptic television series, The Last Ship.

In 2017, she played Charlotte in the fourth season of The Strain TV Series.