The in vitro antioxidant and antimicrobial activities of two Achillea millefolium (Adanson) Koch s.l species essential oils (A. collina Becker ex Heimerl s.l. and A. pannonica Scheele, Asteraceae) originating from the Golija and Radan mountains (Serbia) were investigated. The chemical profiles of the essential oils were evaluated by GC-MS. Antioxidant activity was assessed as free radical scavenging capacity (RSC) towards 2,2-diphenyl-1-picrylhydrazil (DPPH) radicals, together with effects on lipid peroxidation (LP). Antibacterial activity was examined on 21 bacterial strains. Based on the chemical composition of the essential oil, A. collina s.l. from Mount Golija was classified as a chamazulene chemotype (tetraploid). The high percentage of oxygenated monoterpenes and absence of azulene in the essential oil obtained from A. pannonica from Radan pointing that this population is octaploid. Essential oil of A. pannonica expressed stronger antimicrobial activity on almost all tested bacteria. Furthermore, this essential oil expressed higher scavenging effects on DPPH radical (IC(50) = 0.52 comparing to 0.62 mug/mL). Only in the LP evaluation, essential oil of A. collina s.l. from Golija exhibited stronger antioxidant activity (IC(50) = 0.75 comparing to 2.12 mug/mL).