Introduction: Cutaneous Leishmaniasis (CL) is a parasitic disease caused by Leishmania species. Currently accessible treatments remain insufficient, and there is pressure to develop suitable and effectual options. In this study, we used different concentrations of podophyllin in vitro on leishmania parasites and then on leishmaniasis lesions in mice and compared their efficacy. Method: We used podophyllin (14.3 µg/ml) in vitro against leishmania major parasites, then in experimental animals in different concentrations. Results: Podophyllin (14.3 µg/ml) that used in vitro eradicated leishmania major parasites, but, in mice after four weeks was not effective and the diameter of the lesions increase with use of topical podophyllin. Conclusion: Despite the lethal effect on leishmania in vitro, treatment with different doses of podophyllin could not accelerate the healing process of the leishmaniasis lesions of the experimental rats.