In this study, an antibacterial electrospun nanofibers for wound dressing application was successfully prepared from polyvinyl alcohol (PVA), Pluronic F127 (Plur), polyethyleneimine (PEI) blend solution with titanium dioxide nanoparticles (TiO2 NPs). PVA-Plur-PEI nanofibers containing various ratios of TiO2 NPs were obtained. The formation and presence of TiO2 in the PVA-Plu-PEI/ TiO2 composite was confirmed by X-ray diffraction (XRD). Transmission electron microscopy (TEM), Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), mechanical measurement, and antibacterial activity were undertaken in order to characterize the PVA-Plur-PEI/TiO2 nanofiber morphology and properties. The PVA-Plu-PEI nanofibers had a mean diameter of 220 nm, and PVA-Plur-PEI/TiO2 nanofibers had 255 nm. Moreover, the antimicrobial properties of the composite were studied by zone inhibition against Gram-negative bacteria, and the result indicates high antibacterial activity. Results of this antibacterial testing suggest that PVA-Plur-PEI/TiO2 nanofiber may be effective in topical antibacterial treatment in wound care; thus, they are very promising in the application of wound dressings.