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Leishmaniasis is one of the biggest concerns of the World Health Organization (WHO) due to its severe side effects, the emergence of resistance, and secondary bacterial infections; therefore, it was reported as an epidemic, especially in immunocompromised individuals. The main purpose of this study was to synthesize and characterize the polylactic acid (PLA)/chitosan nanofibers decorated with amphotericin B as a new delivery system for the in vitro treatment of leishmaniasis wounds. The prepared nanofibers were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), X-ray diffraction (XRD), and in vitro drug release test. The anti-leishmanial effect of nanofibers decorated with amphotericin B against Leishmania major promastigotes and its cytotoxicity to macrophages were determined by the flow cytometry test. The in vitro drug release assay indicated that 84% of the amphotericin B loaded in nanofibers was released after 400 min. The average concentration of the amphotericin B loaded in PLA/chitosan nanofibers and the conventional form of amphotericin B that prevented the growth of 50% of the promastigotes of L. major were 1.29 and 4.34 µg/mL, respectively.