Objective: To develop and characterize innovative vaginal dosage forms for the treatment of bacterial vaginosis (BV). Significance: This study is the first comparative evaluation of the metronidazole (MET)-loaded polyvinylpyrrolidone (PVP) nanofiber formulations on BV treatment. Vaginal nanofibers are one of the potential innovative dosage forms for BV treatment because of their flexible, mucoadhesive, and easy application in vaginal application which can be applied by the mucosal route. Methods: Blank and MET-loaded PVP solutions were prepared at three different concentrations (10, 12.5, 15%) for produce nanofiber. The suitability of the viscosities, surface tensions, and conductivity values of the solutions used to produce nanofibers for the electrospinning process has been evaluated. Scanning electron microscopy, mucoadhesion, permeability, Fourier transform infrared spectroscopy, differential scanning calorimetry, and drug release tests were performed to reveal the physical, chemical, and pharmaceutical properties of the nanofibers. Mechanical properties, and contact angle of the fibers were also determined. Gel and solution formulations containing MET were prepared for comparative studies. Results: All polymer solutions were found to be suitable for electrospinning process. PVP concentration directly affected nanofiber diameter, mechanical, and mucoadhesion properties of nanofibers. The release profiles of the drug from the nanofibers were similar for all concentration of PVP and release from the fibers was rapid. The permeability coefficient of MET from nanofibers was increased more than gel and solution formulations. Conclusions: Vaginal use of MET-loaded nanofibers has been shown to be a potential drug delivery system for the treatment of BV.