Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram-positive strains tested with different MIC values (0.25-2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram-negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram-positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.