Zoonotic cutaneous leishmaniasis caused by Leishmania major is a most common type of vector-borne disease in Iran. The pentavalent antimonial drugs have been used in the treatment of cutaneous leishmaniasis for a long time, but drug resistance and some of serious side effects have been reported. Thus, discovery and development of new therapeutic candidates are needed. The CM11 peptide is one of these peptides that its anti-bacterial activity has been proven. This peptide is a short cecropin–melittin hybrid peptide obtained through a sequence combination approach. The aim of this study was to evaluate in vitro anti-leishmanial activity of CM11 peptide against amastigote forms of Leishmania major. In this study, amastigote forms of Iranian strain of L. major (MRHO/IR/75/ER) were cultured in the presence of different concentrations of meglumine antimoniate (Glucantime®) to find the most appropriate in vitro concentration of Glucantime® against L. major amastigotes. Then, the anti-leishmanial activities of various concentrations of CM11 peptide (8, 16, 32 and 64 µM) were evaluated for 24, 48 and 72 hr by DAPI staining. In addition, MTT assay was used to determine the cytotoxic effects of CM11 peptide on murine fibroblast cell line. The results showed that CM11 peptide has antimicrobial activity against Iranian isolate of L. major in the laboratory conditions. It seems that the CM11 peptide has significant potential to be used as a new anti-leishmanial agent.