Background: Today, most patients with cutaneous leishmaniasis are treated with drugs such as glucantime; however, severe drug resistance to these drugs is found, in addition to recurrence, extensive drug complications, and secondary infections in many patients. Objectives: Therefore, due to the lack of sufficient information on this subject, this study was undertaken for the first time to investigate the antimicrobial activity of multifunctional lactoferrin as one of the most important bioactive compounds on amastigotes and promastigotes forms of Leishmania major in vitro. Methods: In this study, six concentrations of lactoferrin (2. 5, 5, 10, 20, 40, and 80 g/mL) were added to parasite cultures at specified times. The MTT was done. Leishmania major promastigotes were incorporated to J774 cells and then incubated for 72 hours. The results were compared with the results of glucantime, as a standard. In the end, flow cytometry was performed to investigate apoptosis. Results: After 24, 48, and 72 hours of incubation in the presence of various concentrations of lactoferrin, the number of amastigotes and promastigote parasites increased over time without any significant difference compared to the control group (P > 0. 05), but it was significant compared to glucantime at the 80 g/mL concentration (P < 0. 05). Besides, lactoferrin showed very low toxicity effects on macrophages and stimulated the growth of both forms of L. major parasite. Conclusions: Our findings indicated that lactoferrin could not induce early and late apoptosis in both forms of L. major.