Cucurbita Pepo (Pumpkin) is a plant which is traditionally used to treat a wide variety of diseases, and through scientific investigation most of the properties have been validated. However, more scientific data is needed to support the various health claims. The aim of this study was to determine the fatty acids (FA) and Triacylglycerols (TAG) composition, tocopherols content and to evaluate the antioxidant activity of eight pumpkin seeds oil cultivated in Algeria. The results indicated that pumpkin seeds were rich in oil (15.8–33.5%) and the major unsaturated fatty acids were linoleic acid (42.1–48.5%) followed by oleic acid (18.4–39.6%), while the main saturated fatty acids were palmitic (13.91–20.00%). Unsaturated FA showed a preference for the internal position. Linoleic and oleic acids occurred predominantly in the sn-2 position as generally found in vegetable oils. The main TAGs were triunsaturated GU3 and monosaturated GSU2 as majorities compounds. The tocopherol content of the oils ranged from 7.7 to 31.9 mg/kg of oil for α-tocopherol, from 39.3 to 155.1 mg/kg for (β+γ)-tocopherol, and from 39.0 to 103.0 mg/kg for δ-tocopherol. The capacity that would be suitable for food and industrial applications, as well as high unsaturation FA and tocopherol content that could potentially improve the nutrition of human diets. The data of antioxidant power determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and phosphomolybdenum (PPM) complex methods show that the level of the antioxidant activity by two used assays was significantly compared to synthetic antioxidants. Also, it was demonstrated for the first time that the studied oils possessed a good antioxidant activity which may be associated with their alleged health benefits.