A viable alternative for the next generation of wound dressings is the preparation of electrospun fibers from biodegradable polymers in combination with inorganic nanoparticles. A poly(vinyl alcohol)-chitosan-silver nanoparticles (PVA-CTS-Ag NPs) system has been developed for antimicrobial and wound healing applications. Here, the preparation of PVA-CTS-Ag electrospun fibers using a two-step process is reported in order to analyze changes in the microstructural, mechanical, and antibacterial properties and confirm their potential application in the biomedical field. The Ag nanoparticles were well-dispersed into the chitosan matrix and their cubic structure after the electrospinning process was also retained. The Ag NPs displayed an average diameter of ~33 nm into the CTS matrix, while the size increased up to 213 nm in the PVA-CTS-Ag(NPs) fibers. It was observed that strong chemical interactions exist between organic (CTS) and inorganic phases through nitrogenous groups and the oxygen of the glycosidic bonds. A defect-free morphology was obtained in the PVA-CTS-Ag NPs final fibers with an important enhancement of the mechanical properties as well as of the antibacterial activity compared with pure PVA-CTS electrospun fibers. The results of antibacterial activity against E. coli and S. aureus confirmed that PVA-CTS-Ag(NPs) fibers can be potentially used as a material for biomedical applications.