Polyurethane/polyvinylpyrrolidone/silk nanofibers were prepared by electrospinning. Highly lipophilic antifungal drug sertaconazole nitrate was incorporated into the nanofibers, and drug release profiles were compared with polyurethane/polyvinylpyrrolidone nanofiber. Nanofibers were characterized in terms of morphology, polymer-polymer and drug-polymer compatibility, loading efficiency, surface roughness profile, and hydrophilicity by scanning electron microscopy, Fourier transform infrared spectroscopy, atomic force microscopy, optical tensiometer and high-performance liquid chromatography. In vitro release data revealed that nanofibers fabricated with silk fibroin either as polymer blend or coating material showed drug release in sustained manner. Activity and biocompatibility study analysis data proved that all sertaconazole incorporated nanofiber formulations were fungistatic against Candida albicans and had good biocompatibility, which could be considered as potential topical drug delivery systems used in the treatment of fungal infections as a scaffold applied topically.