Electrospun nanofibrous membranes are of great interest for tissue engineering, active material delivery, and wound dressing. These nanofibers possess unique three-dimensional (3D) interconnected porous structures that result in a higher surface-area-to-volume ratio and porosity. This study was carried out to prepare nanofibrous membranes by electrospinning a blend of PVA/chitosan polymeric solution functionalized with different ratios of copper oxide. Chitosan-stabilized CuO nanoparticles (CH-CuO NPs) were biosynthesized successfully utilizing chitosan as the capping and reducing agent. XRD analysis confirmed the monoclinic structure of CH-CuO NPs. In addition, the electrospun nanofibrous membranes were UV-crosslinked for a definite time. The membranes containing CH-CuO NPs were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectrophotometry, and dynamic light scattering (DLS). SEM results showed the nanosize of the fiber diameter in the range of 147-207 nm. The FTIR spectroscopy results indicated the successful incorporation of CH-CuO NPs into the PVA/chitosan nanofibrous membranes. DSC analysis proved the enhanced thermal stability of the nanofibrous membranes due to UV-crosslinking. Swelling and degradation tests were carried out to ensure membrane stability. Greater antimicrobial activity was observed in the nanoparticle-loaded membrane. An in vitro release study of Cu2+ ions from the membrane was carried out for 24 h. The cytotoxicity of CH-CuO NP-incorporated membranes was investigated to estimate the safe dose of nanoparticles. An in vivo test using the CH-CuO NP-loaded PVA/chitosan membrane was conducted on a mice model, in which wound healing occurred in approximately 12 days. These results confirmed that the biocompatible, nontoxic nanofibrous membranes are ideal for wound-dressing applications.