Rhona Mitra is the daughter of Nora and Anthony Mitra. Her mother is Irish Catholic and her father is of half-Bengali Indian and half-English descent. She was raised as a Roman Catholic. In 1984, Mitra's parents divorced and, as part of an overall lifestyle change, eight-year-old Mitra was sent off to boarding school. Several years passed at two different all-girls schools, including Roedean, but Mitra was eventually expelled from both of them.

She has been quoted as saying: "I was kicked out [of Roedean] for taking a sixth-former's car and driving it down to the local boys' school. I was only 14. I left stuffing under my bedsheets but one of my friends told on me and the headmistress tracked me down. They put me in this room with bars on the windows to punish me. I was stuck in there for a whole week with just a rosary for comfort, having my dinner brought in on trays. The only time I got out was to say confession to the school priest. After that, they booted me out.

Then at the next school the other girls used to blame me whenever they got caught for something, so all the parents wrote in and said they didn't want me at the school. I crammed my exams in London and did fine."

She appeared in the final season of The Practice playing Tara, and continued that role into its spin-off Boston Legal, but left not long into the second season. She also appeared as a live action model for Lara Croft from Eidos Interactive's Tomb Raider video game series. She had a recurring role during Season 3 of Nip/Tuck.

After becoming the official living embodiment of the video game character Lara Croft, she underwent breast implant surgery in an effort to properly "become" the fictional character.

The surgery was performed by her father, a renowned cosmetic surgeon.