In recent years, production of nano-sized wound dressing materials from biopolymers by electrospinning method has attracted great attention. In this study, chitosan that is an inherently antimicrobial polymer and chitosan (CS)/poly(ethylene oxide) (PEO) blend solutions were electrospun to obtain nanofiber webs for wound-dressing applications. Although electrospinning of chitosan with PEO was previously studied, this study emphasized the effect of molecular weight of polymer used, total polymer concentration and electrical conductivity of polymer solution on fiber formation. Results show that selection of suitable solvent and determination of optimum process parameters were key factors for obtaining uniform nanofibers. Antimicrobial activities of these nanofiber webs against S.aureus, E.coli, and C.albicans were tested according to AATCC 100–2004. Over 99% reduction in the number of microorganisms with pure chitosan nanofibers and over 50% reduction with CS/PEO blend nanofibers were obtained.