This study aims to comprehensively examine the chemical composition, physicochemical characterization, as well as antioxidant and antiplatelet activities of Pistacia lentiscus fruit oil (PLFO). Our results showed that the prominent class of fatty acids was represented by monounsaturated fatty acids (42.73%), followed by saturated fatty acids (33.99%) and polyunsaturated fatty acids (23.13%). The different ratios of fatty acids were determined (PUFA/MUFA = 0.54, PUFA/SAFA = 0.68 and MUFA/SAFA = 1.25). The obtained results related to saturated and polysaturated fatty acids ratios were in line with the recommendations of the World Health Organization (WHO). The principal fatty acid (FA) consisted in oleic acid (41.32%), followed by linoleic acid (23.08%). A comparison was made with Moroccan and Algerian PLFO showing that Tunisian PLFO is the richest in linoleic acid. The main tentatively identified triacylglycerols, which are in monounsaturated and polyunsaturated forms, were SLL+PLO (19.73%), OOL+LnPP (13.12%) and POO (10.57%). The primary sterol identified in PLFO was β‐sitosterol (80.19%). However, tocopherols were quantified at a content of 1249.16 mg g–1 of oil. The effect on platelet aggregation was evaluated on human platelet‐rich plasma treated with various PLFO concentrations, dissolved in ethanol (named POE) and then induced by ADP, collagen, and arachidonic acid (AA). The outcome revealed that 8 µL mL–1 of POE strongly inhibited ADP‐induced platelet aggregation in humans. Additionally, the expression of CD63 and P‐selectin as markers of platelet secretion, and αΙΙβ3 integrin activation were assessed by flow cytometry. It has been found that PLFO significantly reduced platelet activation as well as alpha and dense granule secretion. Moreover, the cytotoxicity assay on normal HEK‐293 cells, the hemolysis test and the platelet viability confirmed that PLFO is a safe substance. These findings are valuable for assessing the nutritional composition of PLFO within a preventive and/or therapeutic framework in a clinical context.Practical applications: PLFO is mainly used to alleviate problems related to poor blood circulation, especially for women but it is also employed to treat problems such as heavy legs and varicose veins. Furthermore, PLFO would prevent certain diseases such as phlebitis, and alleviates various disorders, including cardiovascular problems.