The aim of this study was to develop polymeric nanofibers for controlled administration of Amphotericin B (AmpB), using the solution centrifugation technique, characterizing its microstructural and physical properties, release rate, and activity against Leishmania and Candida species. The core-shell nanofibers incorporated with AmpB were synthesized by Solution Blow Spinning (SBS) and characterized by scanning electron microscopy (SEM), differential scanning calorimetry, X-Ray diffraction, and drug release assay. In vitro leishmanicidal and antifungal activity were also evaluated. Fibrous membranes with uniform morphology and smooth surfaces were produced. The intensity of the diffraction peaks becomes slightly more pronounced, assuming the increased crystallization in PLA/PEG at high AmpB loadings. Drug release occurred and the solutions with nanofibers to encourage greater incorporation of AmpB showed a higher concentration. In the results of the experiment with promastigotes, the wells treated with nanofibers containing concentrations of AmpB at 0.25, 0.5, and 1%, did not have any viable cells, similar to the positive control. Various concentrations of AmpB improved the inhibition of fungal growth. The delivery system based on PLA/PEG nanofibers was properly developed for AmpB, presenting a controlled release and a successful encapsulation, as well as antifungal and antileishmanial activity.