Abstract Pomegranate seeds are an excellent source of compounds with biological activity. In the present study, the pomegranate seed oil (PSO) was tested for antioxidant and cytotoxic potential. PSO was extracted by supercritical fluid extraction with previous treatment of seeds by microwaves and analysed for its fatty acid profile, carotenoid and tocopherol contents. PSO demonstrated to be a good source of polyunsaturated fatty acids (PUFAs) (83.5%), followed by monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA) (9.5% and 7.0%, respectively). Concerning fatty acid profile, punicic (C18:3), oleic (C18:1) and linoleic (C18:2) acids were the predominant ones. Carotenoids and tocopherols were identified and quantified by HPLC. Results showed that the PSO was a good source of γ- tocopherol as dominant among investigated compounds (128.6 mg/100 g). The extract was screened for its antioxidant activity using α-TEAC assay. Cytotoxic effect was determined by MTT test against malignant HeLa, LS174 and A549 cells (IC50 = 49.51–91.54 μg/mL), and against normal MRC-5 and EA.hy926 cells (IC50 > 200 μg/mL). The best effect was exhibited on cervix adenocarcinoma cell line while no cytotoxicity was found for normal cells pointing out PSO safety.