This study aims to characterize and evaluate polyethylene oxide (PEO) and chitosan (CS) nanofibers produced by electrospinning method. Electrospinning solutions were used at three different concentrations (1, 2, 3 wt%) with five different PEO/CS mixing ratios (30/70, 40/60, 50/50, 60/40, 100/0). FESEM, XRD and FTIR tests were applied for characterization of the nanofibers. Antibacterial activity of the nanofibers against Staphylococcus aureus and Klebsiella pneumoniae microorganisms was investigated using disk diffusion method. While 1 wt% of concentration was not suitable to obtain regular nanofibers, the nanofibers were uniform and largely free of beads at the other ones (2, 3 wt%). The average diameters of the nanofibers varied from 59 to 298 nm depending on the concentration and mixing ratio. Strong hydrogen bonds were formed between two polymers, while the crystal structure of PEO did not change significantly when mixed with chitosan. The PEO/CS nanofibers showed no resistance to the selected bacteria.