The present study reports the chemical composition of headspace volatiles (HS) and acetone extracts of the endemic Bulgarian species Achillea thracica Velen. from its natural habitat (N), in vitro propagated (IN) and ex vitro established (EX) plants. Additionally, acetone extracts were tested by a disk diffusion method for antibacterial activity. Irregular monoterpenes were the most abundant HS volatile constituents, while 0,0-dimethyl quercetin was the most abundant flavonoid in the acetone extracts. The secondary metabolites of A. thracica grown in its natural habitat (N), propagated in vitro (IN) and ex vitro established (EX) showed that the qualitative composition is mutually similar, but there are differences in the quantitative composition. Considering antibacterial activity, IN and EX samples showed moderate activity against Pseudomonas aeruginosa and Escherichia coli.