Context: Natural products and their compounds are some of the most interesting sources of new drugs. Reviews have reported various pharmacological properties such as antimicrobial effects of Zataria multiflora Boiss (Lamiaceae). Objective: The present study investigates the chemical composition of Z. multiflora essential oil and evaluates its cytotoxic effects and anti-leishmanial activities against Leishmania tropica in an in vitro model. Materials and methods: The components of Z. multiflora oil were identified by gas chromatography/mass spectroscopy (GC/MS) analysis. Anti-leishmanial effects of the essential oil (0–100 μL/mL) and methanol extract of Z. multiflora (0–100 μg/mL) on promastigote forms as well as their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 h. The leishmanicidal activity against amastigote forms of L. tropica was evaluated at the concentrations of 0–50 μg/mL in a macrophage model for 48 h. Results: The chemical analyses demonstrated that the main components of essential oil were thymol (41.81%), carvacrol (28.85%), and p-cymene (8.36%). Regarding leishmanicidal activity, the IC₅₀ values for the essential oil and methanol extract were 3.2 μL/mL and 9.8 μg/mL against promastigote forms and 8.3 μL/mL and 34.6 μg/mL against amastigote forms, respectively. Essential oil (CC₅₀ 89.3 μL/mL) indicated a higher cytotoxic effect than the methanol extract (CC₅₀ 591.6 μg/mL) of Z. multiflora. Conclusion: The present study revealed the chemical composition of Z. multiflora that might be a natural source of new anti-leishmanial agents in terms of use against cutaneous leishmaniasis.