Barbara Lukešová graduated from the State Conservatory in Prague. After that she acted at the F. X. Šalda Theatre in Liberec and later with the Rokoko Theatre in Prague (1994-2001). At the same time, she was performing at the Theatre in Celetna. She also starred in various productions of the Rubín Theatre, National Theatre, Prague Municipal Theatres, Divadlo Komedie and the Theatre in Řeznická.