Continuous defect-free nanofibers containing chitosan (Ch) or quaternized chitosan (QCh) were successfully prepared by one-step electrospinning of Ch or QCh solutions mixed with poly[(L-lactide)-co-(d,l-lactide)] in common solvent. XPS revealed the surface chemical composition of the bicomponent electrospun mats. Crosslinked Ch- and QCh-containing nanofibers exhibited higher kill rates against bacteria S. aureus and E. coli than the corresponding solvent-cast films. SEM observations showed that hybrid mats were very effective in suppressing the adhesion of pathogenic bacteria S. aureus. The hybrid nanofibers are promising for wound-healing applications.