<p>This study aimed to develop and evaluate nanofiber scaffolds for advanced wound dressing applications. The nanofibers were fabricated using polyvinyl alcohol, sodium polyacrylic acid, and graphene oxide and were systematically assessed for their physical, chemical, and biological properties. Morphological analysis through scanning electron microscopy revealed a uniform and homogeneous nanofiber structure, while tensile testing confirmed their excellent mechanical strength. Crystallographic characteristics were examined using X-ray diffraction, and Fourier Transform Infrared Spectroscopy verified the chemical composition. The nanofibers demonstrated remarkable fluid absorption capabilities, accommodating both water and blood, which are crucial for effective wound management. Tests for water vapor porosity and permeability further highlighted their ability to create an optimal environment for wound healing. Biological evaluations, including hemolysis and blood adaptation tests, confirmed the nanofibers’ biocompatibility. Antibacterial efficacy was thoroughly assessed using MIC, MBC, anti-biofilm, and bacterial penetration tests, showcasing significant inhibitory effects on bacterial activity. Cytotoxicity analysis (MTT) and nuclear staining (DAPI) revealed low toxicity and excellent biocompatibility. Additionally, scratch wound assays demonstrated the positive impact of the nanofibers on cellular migration and signaling, vital for tissue regeneration. In vivo experiments and histological analyses provided further evidence of the nanofibers’ ability to support tissue repair and regeneration.</p>