Amphotericin B (AmB) is an antileishmanial drug with high toxicity; however, this drawback might overcome by decreasing the AmB self-aggregation state. This work aimed at evaluating the influence of cholesterol on the aggregation state of AmB loaded in a nanoemulsion (NE-AmB) for the treatment of cutaneous leishmaniasis. NE-AmB (1, 4 and 8 mg/kg/day) was administered intravenously to animals infected by every 2 days for a total of five injections. Ultraviolet-visible spectroscopy and circular dichroism studies demonstrated that cholesterol reduced AmB aggregation state in NE. NE-AmB was stable after 180 days, and its hemolytic toxicity was lower than that observed for the conventional AmB. NE-AmB administered intravenously into animals infected by at 8 mg/kg was capable of stabilizing the lesion size and reducing the parasitic load. These findings support the NE potential as a stable nanocarrier for AmB in the treatment of cutaneous leishmaniasis.