Leishmaniasis is a neglected infectious disease caused by protozoan parasites from Leishmania genus species, affecting millions of people, in several countries. The current available treatment for cutaneous leishmaniasis (CL) has presented many side effects. In this way, micro- and nanotechnology are important processes, since they may be useful for release profile modulation of CL drugs improving their bioavailability. Amphotericin B (AmB) is a macrolide antibiotic used as a second-choice treatment. This study aimed the development of oil-water nanoemulsions (NEs) containing AmB for topical administration to treat CL. Furthermore, NEs were characterized by their droplet size, morphology, drug content, stability, in vitro release profile, and ex vivo skin permeation. In vitro anti-leishmanial activity using Leishmania amazonensis promastigotes was also evaluated. NEs containing AmB presented droplet size lower than 60 nm with a polydispersity index lower than 0.5. The best AmB-NEs were submitted to stability tests and these formulations presented excellent results after 365 days under refrigeration, confirming the maintenance of the drug content higher than 95%. AmB-NEs displayed slow and controlled AmB kinetic release and low skin permeation. These formulations presented lower cytotoxicity in comparison with free AmB and higher anti-leishmanial effect against L. amazonensis promastigotes. Therefore, the selected AmB-NE formulations, especially AmB-NE01, presented promising results as novel alternatives for CL treatment. Graphical abstract.