Leishmaniosis caused by Leishmania major is one of the main infectious diseases that infected populations in developing countries around the world. We assessed the effectiveness of berberine chloride nanoliposomes (BcNLs) against L. major promastigotes in vitro. Nanoliposomal berberine chloride was prepared using thin film hydration method and characterized based on encapsulation efficiency, size and zeta potential. Anti-Leishmania effect of different concentrations (0.05-60 μg/ml) of BcNLs as studied in L. major (MRHO/IR/75/ER) at 24, 48 and 72 h using the hemocytometer technique. Berberine chloride was successfully loaded into nanoliposomes with encapsulation efficiency of 85.54%. The surface charge of nanoparticle is neutral and the morphology of nanoliposomal berbrine chloride is spherical without any agglomeration. Cell viability assay was performed on HFF cell line to show biocompatibility of liposome nanoparticles. IC50 of BcNPs at 24, 48 and 72 h against L. major were found to be 7.6, 5.96 and 3.19 μg/ml, respectively. BcNLs showed a significant anti-Leishmania effect and induced a better and more tangible effect on the survival of L. major promastigotes and could be suitable candidates for further investigation. The results showed that the BcNLs agent is effective against L. major promastigotes and may be a promising alternative to current treatments.