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This study aimed to assess the antileishmanial activity of biomolecules obtained from Oleaeuropaea L. leaves and twigs recovered from eight Tunisian cultivars. The extraction was first carried out with 80% methanol, and then the obtained extract was fractionated using three solvents of increasing polarity: cyclohexane (CHX), dichloromethane (DCM) and ethyl acetate (EtOAc). The antileishmanial activity was determined against leishmanial strains responsible for cutaneous, visceral, and mucocutaneous leishmaniasis. The cyclohexane fraction of the leaves of cv. Chemlali from the region of Sidi-Bouzid exhibited the strongest leishmanicidal activity against all the tested leishmanial strains. The inhibition concentrations (IC50) were 16.5, 14.5, and 7.4 μg mL−1 for Leishmaniamexicana (cutaneous), Leishmaniabraziliensis (mucocutaneous), and Leishmaniadonovani (visceral), respectively. Interestingly, low cytotoxicity was observed on THP-1 cells with selective indexes (SI) ranging from 22.8 to 50.5. HPLC-HRMS and full-house NMR analysis allowed the identification of three triterpenic compounds, oleanolic acid (IC50 = 64.1 μg mL−1), erythrodiol (IC50 = 52.0 µg mL−1), and uvaol (IC50 = 53.8 μg mL−1). Antileishmanial activity of uvaol and oleanolic acid has been previously reported. However, this work constitutes the first report of the antileishmanial activity of erythrodiol which showed combinatorial interaction with uvaol (IC50 = 26.1 μg mL−1) against Leishmaniatropica. The mixture of the three compounds, as major ones, exhibited an enhanced activity against Leishmaniatropica (IC50 = 16.3 µg mL−1) compared to erythrodiol alone or the combination of uvaol and erythrodiol. This finding is of great importance and needs further investigation. [ABSTRACT FROM AUTHOR]