Pumpkin has ever been applied in the food industry for the production of purees, juices, jams and alcoholic beverages generating waste in the form of rind and seeds. The seeds are used for both edible and medicinal purposes. In fact, pumpkin seed oil has been reported to possess a strong antioxidant potential and has been identified as an exceptional preventive against hypertension and carcinogenic diseases (Rezig et al., 2012). Pumpkin seeds are also used in culinary practices mainly in the southern parts of Austria, Slovenia, and Hungary (Murkovic et al., 1996). Pumpkin seeds are also consumed after being salted and roasted in African countries, mainly in Tunisia. The presentation will concern the evaluation of the bioactive compounds of pumpkin (Cucurbita pepo) seed oil of the ‘Essahli’ Tunisian cultivar following a cold pressed extraction method. Gas chromatography revealed that the major fatty acids were linoleic, oleic, and palmitic acids (45,90%, 29,3%, and 15,65% respectively). The Seed oil was also found to be rich in sterols (4849.62 mg/kg oil). The sterol marker β-sitosterol accounted for 39.66 % of total sterols contained in the seed oil of this variety. Total phenolic and flavonoids contents of Cucurbita pepo var. ‘Essahli’ were respectively 41.24 mg GAE/100g and 8.24 mg rutin equivalent/100g. IC50 of the pumpkin seed oil was of 64.71 µg/g. The oil stability index was of 3.74h. Pumpkin seed oil revealed the presence of many bioactive substances such as flavonoids, total phenolic content, and phytosterols. These compounds may be used as natural antioxidants for industrial applications. Besides, the high-unsaturated fatty acid level makes the melon seed oil nutritionally valuable. Therefore, pumpkin seeds are of great interest thanks to their bioactive molecules and their plant oil. Thus, appropriate use of pumpkin seeds makes them a good example for valorization of vegetable processing by-products.