Chitosan (CS) blended with poly(ethylene oxide) (PEO) was electrospun into nanofibrous mats. The spinning solution of 6.7 : 0.3 (% w/v) of CS : PEO was dissolved in a 70 : 30 (v/v) trifluoroacetic acid/dichloromethane solution. The obtained fibers were smooth without beads on their surfaces and average diameter of the fiber was 272 ± 56 nm. N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTACC) and N-benzyl-N,N-dimethyl chitosan iodide (QBzCS) were each prepared from the CS/PEO mats. They were identified by Fourier-transform infrared and X-ray photoelectron spectroscopy and degree of swelling in water. Both quaternized electrospun chitosan mats exhibited superior antibacterial activity to the unmodified electrospun CS/PEO against Staphylococcus aureus and Escherichia coli at short contact times. After 4 h of contact, the reduction of both bacterial strains by CS/PEO, HTACC, and QBzCS was equal at about 99–100%. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40981.