AbstractElectrospun nanofibers are unique one‐dimensional materials with high surface area to volume ratio which can be used in various applications including in antibacterial application. In this study, we demonstrated a simple fabrication of polyethylene oxide‐polypyrrole (PEO‐PPy) composite nanofibers with antibacterial properties. The polymerization of pyrrole was conducted using ammonium persulfate (APS) initiator at ambient temperature for 24 h. The resulting PEO‐PPy nanofibers were then characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), fourier transform infrared (FTIR), and tensile tests. The morphology of nanofibers observed by SEM revealed that the minimum concentration of PEO was 8 wt% to obtain the bead‐free nanofibers. Meanwhile, variation of pyrrole concentration does not alter the morphology of nanofibers significantly. The crystallinity of the nanofibers increased with increasing the PEO amount indicated by two typical peaks located at 2θ=19° and 23°, respectively. On the other hand, polypyrrole contributed the antibacterial properties significantly to the nanofibers and as a result, the antibacterial activity determined by UV‐Vis spectrophotometer increased dramatically with increasing pyrrole concentration from 0.1 to 0.2, 0.3, 0.4, and 0.5 mM. The electrospun nanofiber mats exhibited good antibacterial properties onE. coliand biocompatibility properties on L929 fibroblasts cell.