The aim of this work was to develop and characterize nisin-loaded polyethylene oxide (PEO) nanofibers prepared by green electrospinning. The electrospinning operating parameters were optimized and nanofibers with 20,000 and 30,000 IU mL-1 of nisin were obtained. Scanning electron microscopy (SEM) imaging confirmed the formation of uniform, beadless nanofibers with diameters that ranged between 216 and 261 nm. Thermal stability studies showed that the electrospinning process led to the impairment of the crystalline structure of the polymer. Also, Raman spectroscopy tests revealed the interaction between nisin and PEO, while attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) showed that the addition of nisin did not alter the polymer backbone structure. In addition to this, the X-ray diffraction (XRD) analysis confirmed the semi-crystalline structure of nanofibers. Finally, the resulting nisin-loaded PEO nanofibers were tested against Staphylococcus aureus, confirming its antibacterial activity against this Gram‑positive bacteria. Moreover, we also report, apparently for the first time, the antibacterial activity of nisin-loaded PEO nanofibers against Bacillus subtilis. These results expand their applications as green antibacterial biomaterials in areas such as food systems and biomedicine.