Background: Although a majority of patients with cutaneous leishmaniasis (CL) are healed with Glucantime chemotherapy, the increased drug resistance rate following its consumption is a concern. In this study , Naja naja oxiana crude venom of cobra snakes was used for the first time as an assembled combination of bioactive pharmaceutical components on Leishmania major (L. major) standard strain . Objective: To evaluate the efficacy of the Naja naja oxiana crude venom of Iranian cobra snakes on Leishmania major standard strain in vitro. Methods: Five concentrations (1.25, 2.5, 5, 10 and 20 µg/mL) of venom were added to Leishmania major cultures at 24, 48 and 72 h. The viability of the parasites and venom toxicity were assessed by MTT test. The apoptosis was determined by flowcytometry, while IC50 was determined by counting parasites compared to that of glucantime. Each test was conducted in triplicate. Results: After exposure the venom at 2.5 µg/mL for 72 h, IC50 value was 0.36 µg/mL and 14.12 µg/mL for promastigotes and amastigotes, respectively. MTT valuation clarified 1.01% promastigotes viability. Furthermore, the results indicated that the venom of Naja naja oxiana induced early and late apoptosis in parasites. Conclusions: The venom of Naja naja oxiana revealed remarkable anti-leishmanial effects as a novel anti-parasitic alternative. Thus the bioactive and effective fractions of this venom may be considered as anti-leishmanial candidates in future studies.