The composition and content of certain bioactive components of the cold pressed oil obtained from six samples of pumpkin seeds (Cucurbita pepo L.) cultivated in Serbia were analyzed by GC and GC/MS. The composition and content of fatty acids, tocopherols and phytosterols, and the total content of squalene were determined. The results indicate oil's excellent quality, with high contents of monounsaturated fatty acids (37.1 ± 0.70–43.6 ± 0.69 g/100 g of total fatty acids), total tocopherols (38.03 ± 0.25–64.11 ± 0.07 mg/100 g of oil), sterols (718.1 ± 6.1–897.8 ± 6.8 mg/100 g of oil) and especially squalene (583.2 ± 23.6–747 ± 16 mg/100 g of oil). High content of squalene, phytosterols and monounsaturated fatty acids recommend the use of this type of the oil in the nutritional and medical purposes.