This study investigated the extraction, characterization, and fabrication of nanofibers from polysaccharides derived from Cordia myxa fruit using hot water extraction (HWE) and ultrasound-assisted extraction (USAE). Extraction conditions were optimized using response surface methodology, yielding 8.93% (HWE) and 8.40% (USAE), which confirmed the efficiency and shorter processing time of USAE. Although both methods produced polysaccharides with similar saccharide compositions, the USAE-derived sample showed superior quality-being protein-free (vs. 8.2% protein in HWE), with lower ash content (4.3%) and stronger antioxidant activity. It also exhibited higher apparent viscosity and greater colloidal stability (−71 mV vs. −56 mV), indicating improved purity and functionality. USAE-derived polysaccharide was selected for nanofiber fabrication. Pure polysaccharide was not electrospinnable, but blending with 6% (w/v) polyvinyl alcohol (PVA) enabled the formation of uniform, bead-free fibers at low polysaccharide concentrations (0.25–0.5% (w/v)). The nanofibers exhibited high porosity, enhanced thermal stability, and strong crosslinking after treatment with glutaraldehyde vapor. They also showed excellent water absorption and slow degradation, making them suitable for wound dressing use. Antioxidant and release analyses revealed complete release of the 0.25% formulation within 72 h, while the 0.5% fibers showed a slower, sustained release profile. Cytocompatibility and adhesion assays using L929 fibroblasts confirmed non-cytotoxic behavior and improved cell spreading on polysaccharide-containing mats. Overall, ultrasound-assisted extraction coupled with PVA electrospinning produced antioxidant, biocompatible, and thermally stable nanofibers with strong potential for wound-healing applications.