Chronic wounds have been a global health risk that demands intensive exploration. A tri-layered biomaterial scaffold has been developed for skin wounds. The top layer of the scaffold is superhydrophobic, and the bottom layer is hydrophilic, both of which were electrospun using recycled expanded polystyrene (EPS) and monofilament fishing line (MFL), respectively. The intermediate layer of the scaffold comprised hydrogel by cross-linking chitosan (CS) with polyethylene glycol. The surface morphology, surface chemistry, thermal degradation, and wettability characteristics of each layer of the scaffold were examined. Also, the antibacterial activity and in vitro cytotoxicity study on the combined tri-layered scaffold were assessed against and . Data revealed exceptional water repellency of the heat-treated electrospun top superhydrophobic layer (TSL) with a high-water contact angle (WCA) of 172.44°. A TSL with 15 wt% of micro-/nano-inclusions had the best thermal stability above 400 °C. The bottom hydrophilic layer (BHL) displayed a WCA of 9.91°. Therapeutically, the synergistic effect of the combined tri-layered scaffold significantly inhibited bacteria growth by 70.5% for and 68.6% for . Furthermore, cell viability is enhanced when PEG is included as part of the intermediate CS hydrogel layer (ICHL) composition.