: Leishmaniasis is a neglected tropical disease caused by a protozoan parasite of . This study aimed to evaluate the in vitro efficacy and toxicity of a previously developed amphotericin gel as a possible treatment for cutaneous leishmaniasis. : First, quality control of the AmB-gel was carried out, including microbiological stability. The permeated and retained drug was tested on healthy and lacerated human skin. Tolerance to the AmB-gel was tested in vitro using HaCaT, RAW 264.7, and J774 cell lines and by an irritation test (HET-CAM). Promastigotes and amastigotes of various species were tested, and the microscopic morphology of promastigotes exposed to the formulation was analyzed. Computational analysis was performed on the drug, polymer, and ergosterol in the promastigote. : The AmB-gel presented appropriate characteristics for topical use, including no microbial contamination after storage. The amount of drug retained on the intact and injured skin was 1180.00 ± 13.54 µg/g/cm and 750.18 ± 5.43 µg/g/cm, respectively. The AmB-gel did not cause significant signs of toxicity. The IC of the AmB-gel for promastigotes was less than 1 µg/mL for the four species examined, i.e., , , , and , and less than 2 µg/mL for amastigotes of and . The AmB-gel caused notable effects on the surface of promastigotes. Computational analysis revealed primarily hydrophobic and van der Waals interactions between AmB and Pluronic F127 and ergosterol. : Based on the drug retention content and IC values observed for both parasite stages, the AmB-gel may be a promising candidate for in vivo studies in patients with cutaneous leishmaniasis.