Mwine was born in Hanover, New Hampshire to Ugandan parents in 1967. His father was a Harvard Law School-educated attorney. His parents separated when Ntare was 7, with Ntare spending time with his father (who was then working in Finance in the United States, including a period at the World Bank in Washington D.C.) and his mother (who went to Kenya to teach psychology at the University of Nairobi).

Mwine began appearing in stage productions in 1992, appearing as the con man posing as Sidney Poitier's son in Six Degrees of Separation, and in The Riddles Of Race, Circa '68 in 1994, In 1992 and 1997, Mwine was nominated for a Helen Hayes Award for Outstanding Lead Actor in a Non-Resident Production, for his role in Six Degrees of Separation at the National Theatre and Nomathemba at the Kennedy Center in Washington, D.C.. He played Julius Van George in Scent of the Roses at the Seattle Contemporary Theatre in 1998.

Mwine has appeared in movies including Blood Diamond, where he made his film debut. His first appearance in television was in New York Undercover, in 1995. Recent appearances include a recurring role as the mysterious Usutu in Heroes. Mwine originally had the role of "Joseph" in the unaired pilot episode of the show; this part was removed when NBC took on the show full-time, due to the character's plot revolving around terrorist activity. Mwine also appeared as Tom Adler in CSI: Crime Scene Investigation and as Maurice Devereaux in The Riches.

Mwine's first effort as a Playwright, a barestage one-man show entitled Biro, about a HIV-positive Ugandan former rebel soldier who enters the United States illegally for treatment. The play, depicting a 90-minute explanation from the eponymous character to his Lawyer about how he came to be in a Texas jail, premiered in early 2003 at Uganda's National Theatre. It later showed at the Joseph Papp Public Theater in New York, as well as in Los Angeles, Seattle, London, and throughout Africa. Mwine performed the work for multiple African heads of state and then-UN General Secretary Kofi Annan in 2004. The Seattle Post-Intelligencer described his performance as "radiant", particularly so given the dark subject matter.

Mwine's inaugural documentary, Beware of Time, screened at the 2004 Pan African Film Festival in Los Angeles and the Black International Cinema in Berlin. Describing the lives of HIV-positive Ugandans, it was named the Best Film on Matters Relating to Marginalized People, and features a rare interview with Amule Amin, brother of former Ugandan dictator Idi Amin.

In 2018, Mwine featured as Ronnie in the Showtime television series The Chi, which follows residents of the Chicago South Side.