The objective of the study was to develop, optimize and evaluate a nanoemulsion (NE) of Amphotericin B (AmB) using excipients with inherent antifungal activities (Candida albicans and Aspergillus niger) for topical delivery. AmB-loaded NE was prepared using Capmul PG8 (CPG8), labrasol and polyethylene glycol-400 by spontaneous titration method and evaluated for mean particle size, polydispersity index, zeta potential and zone of inhibition (ZOI). NE6 composed of CPG8 (15%w/w), S (24%w/w) and water (61%w/w) was finally selected as optimized NE. AmB-NE6 was studied for improved in vitro release, ex vivo skin permeation and deposition using the Franz diffusion cell across the rat skin followed with drug penetration using confocal laser scanning microscopy (CLSM) as compared to drug solution (DS) and commercial Fungisome. The results of in vitro studies exhibited the maximum ZOI value of NE6 as 19.1 ± 1.4 and 22.8 ± 2.0 mm against A. niger and C. albicans, respectively, along with desired globular size (49.5 ± 1.5 nm), zeta potential (-24.59 mV) and spherical morphology. AmB-NE6 revealed slow and sustained release of AmB as compared to DS in buffer solution (pH 7.4). Furthermore, AmB-NE6 elicited the highest flux rate (22.88 ± 1.7 μg/cm/h) as compared to DS (2.7 ± 0.02 μg/cm/h) and Fungisome (11.5 ± 1.0 μg/cm/h). Moreover, the enhancement ratio and drug deposition were found to be highest in AmB-NE6 than DS across the stratum corneum barrier. Finally, CLSM results corroborated enhanced penetration of the AmB-NE6 across the skin as compared to Fungisome and DS suggesting an efficient, stable and sustained topical delivery.