This study aimed to develop nanoemulsions (NEs) for the topical delivery of Amphotericin B using lipids and surfactants with innate antifungal activity. NEs were formulated by a slow spontaneous titration method and characterized for particle size, polydispersity index, zeta potential, zone of inhibition (ZOI), in vitro release, enhanced ex vivo rat skin permeation-deposition, hemolysis followed by interaction with the skin using scanning electron microscopy, and histopathology. The ZOI values of the optimized NEs (ANE3) were 21.8±1.5 and 19.7±1.2 mm against A. fumigatus and C. albicans, respectively. The explored excipients and optimized ANE3 elicited hemo-biocompatibility. ANE3 exhibited in vitro sustained release and an enhanced flux value (21.62±1.6 μg/cm/h) as compared to the drug solution and Fungisome without displaying toxicity. Conclusively, ANE3 could be a promising therapeutic approach with enhanced efficacy and safety for treating a wide range of fungal infections topically.