Amphotericin B (AmB) is an antifungal and antiparasitic agent that is the main drug used for the treatment of mycoses infections and leishmaniasis. However, its high toxicity and side effects are the main difficulties attributed to its application. In this study, to minimize its harmful effects, AmB-loaded core-shell nanofibers were fabricated, using polyvinyl alcohol, chitosan, and AmB as the core, and polyethylene oxide and gelatin as the shell-forming components. The nanofibers were characterized, using scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, tensile test, drug release, and MTT assay. The results showed that the prepared nanofibers were smooth and had a core-shell structure with almost no cytotoxicity against fibroblast cells and the release study suggested that the core-shell structure decreased the burst release. The disk diffusion assay revealed that the nanofibrous mats at different AmB concentrations exhibited significant activity against all the eight evaluated fungal species with the inhibition zones of 1.4-2.6 cm. The flow cytometry assay also showed that the prepared nanofibrous mat significantly killed Leishmania major promastigotes up to 84%. The obtained results indicated that this AmB-loaded nanofibrous system could be a suitable candidate for a topical drug delivery system for the treatment of both superficial mycoses and cutaneous leishmaniasis., (Copyright © 2021 Elsevier B.V. All rights reserved.)