The volatile compounds from Peucedanum cervaria (Lap.) L. were obtained by hydrodistillation (HD) and headspace solid-phase microextraction techniques (HS-SPME), and then analyzed by GC/MS methods. The composition of samples from a botanical garden was compared with plants collected in the wild. The main compounds of the essential oils of P. cervaria were identified as alpha-pinene, sabinene, and beta-pinene (more than 80% of oil). The content of beta-myrcene, limonene+beta-phellandrene, and germacrene D was higher than 1%. The in vitro antibacterial activity of the essential oil was evaluated by the agar dilution method against ten reference strains of Gram-positive and Gram-negative bacteria.