ABSTRACT Leishmaniasis is a neglected parasitic disease. Numerous studies suggested that nanoemulsions could improve drug delivery in the treatment of zoonotic cutaneous leishmaniasis. This study aims to determine the effect of chitosan-coated nanoemulsions of ozonated olive oil on Leishmania major promastigote/amastigote in vitro. Nanoemulsions (NEs) were prepared using a sonicator bath at 37 °C for one hour at 1000 W, followed by 24 hours of stirring at pH 5.5. The NE was then supplemented with chitosan and stirred all night long at a low speed. Droplet size, zeta potential, in vitro drug release, and cytotoxicity were examined to optimize the chitosan nanoemulsions (CNE) and chitosan ozonated nanoemulsions (CONE). CONE and CNE were found to have droplet sizes and zeta potentials of 16.86 nm, 19.03 nm, 19.7 mV, and 19.3 mV, respectively. Additionally, the final CONE’s drug loading and encapsulation efficiency were determined to be 100% and 3.21%, respectively. The lower doses of CONE had no cytotoxic effects on macrophages. Additionally, it has the ability to inhibit L. major promastigote/amastigote growth. Therefore, the lower doses of the CONE formulation can inhibit the growth of promastigote and amastigote of L. major in vitro while not being toxic to macrophages.