Pumpkin seeds are used in the treatment of benign prostatic hyperplasia and the active constituents have been reported to be the sterols and fatty acids. In this study, the oils extracted from the two seed samples of pumpkin (Cucurbita pepo L. ssp. pepo) growing in Turkey were analyzed for their fatty acid contents. Fatty acids were determined by capillary gas chromatography-mass spectrometry (GC-MS) equipped with a flame ionization detector. The results showed that both samples, which had very similar fatty acid compositions, were quite rich in linoleic acid (43.3 and 43.8%), followed by oleic (36.8 and 35.8%), palmitic (11.9 and 10.9), and stearic acids (6.7 and 7.6%). Saponification, esterification, iodine, peroxide, acidification indexes, and unsaponifiable matter of the oils were also calculated. In addition, the two oil samples were tested for their antibacterial, antifungal, and antiviral activities. Both of the oils displayed the same activity profile, having a good antibacterial activity against Klebsiella pneumoniae and Acinobacter baumannii (16 mu g/mL), potent antifungal activity against Candida albicans (8 mu g/mL), as well as a moderate antiviral effect towards Parainfluenza virus type-3 (16-8 mu g/mL).