In this work, nanofibers of chitosan/poly (ethylene oxide) (PEO) with an average fiber diameter from a few microns down to about 30 nm and a narrow size distribution were fabricated by a fast electrospinning process using a handheld electrospinning device. It was found that the matrix with a formulation of chitosan/PEO ratio of 90/10 (w/w) and 0.3% Triton (R) X-100 retained excellent integrity of the fibrous structure in 0.5M acetic acid solution. The characterization of nanofibrous structure by scanning electron microscope (SEM) imaging showed the homogeneity of nanofibrous structure without considerable bead-like structures. The excellent electro spinnability of the current formulation represents electrospinning of natural biopolymer chitosan as a useful process in various biomedical applications, especially as potential wound dressing agents. Non woven mats and polymer solutions with composition chitosan/PEO ratio of 90/10 and 60/40 showed excellent antibacterial activity against E. coli and P. aeruginosa. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 000: 000-000, 2011