This study aimed to produce nanofibers based on poly(vinyl pyrrolidone)/gelatin/thyme essential oil (PVP/GEL/TEO) using oil-in-water emulsions. Polymer solutions and nanofibers were characterized with viscosity, conductivity, surface tension, Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet?visible Spectroscopy (UV?vis), and gas chromatography-mass spectrometry (GC-MS) spectra. Lastly, antibacterial activity tests were carried out by disc diffusion against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Candida albicans (C. albicans), Pseudomonas aeruginosa (P. aeruginosa), and Enterococcus faecalis (E. faecalis). GC-MS revealed carvacrol (69.96%) to be the major component of TEO. Solution conductivity and surface tension decreased with increasing TEO concentration, while viscosity and absorbance values increased. The average fiber diameter increased and presence of beads decreased with TEO concentration increment, but at 5 wt% TEO, a sticky electrospun mat was obtained. PVP/GEL/TEO nanofibers displayed good antibacterial activity, and inhibition zone diameters increased with TEO concentration. Nanofibers stored at 24 ;C and 37 ;C demonstrated antibacterial activity over a period of 192 h.