Novel hybrid nanomaterials have been developed for antimicrobial applications. Here we introduce a green route to produce antibacterial nanofiber mats loaded with silver nanoparticles (Ag-NPs, 25 nm diameter) enveloped in chitosan after reduction with glucose. The nanofiber mats were obtained from colloidal dispersions of chitosan-based Ag-NPs blended with polyvinyl alcohol. Nanofibers (150 nm average diameter and narrow size distribution) were obtained by electrospinning and cross-linked with glutaraldhyde. The effect of crosslinking on the release of silver was studied by atomic absorption spectroscopy. Antimicrobial activity was studied by the viable cell-counting; mats loaded with silver and control samples (chitosan/PVA) with different degrees of cross-linking were compared for their effectiveness in reducing or halting the growth of aerobic bacteria. The results showed superior properties and synergistic antibacterial effects by combining chitosan with Ag-NPs.