Background: Nanostructured wound dressings produced by electrospinning biocompatible polymers possess great potential because they resemble the natural extracellular matrix and support cell adhesion, proliferation, and differentiation. This study seeks to fabricate mupirocin, keratin, and coenzyme Q10 (Co Q10)-loaded PVA electrospun scaffolds intended for wound healing application and to characterize their morphology, physical properties, antibacterial activity, and biocompatibility. Polyvinyl alcohol (PVA) (10% w/v), various concentrations of keratin/Co Q10 fibrous scaffolds (electrospun at a voltage of 50 kV, flow rate of 4 mL/h), and 2% mupirocin was designed and fabricated to activate keratinocytes in the wound bed, stimulate cell proliferation, and increase antimicrobial penetration. Results: The diameters of the scaffolds were observed to be in the nanoparticulate range 2.11 ± 0.20 to 3.27 ± 0.10 nm. By 30 min, all the scaffolds had more than 50% of the cumulative concentration of mupirocin released with 51.06 ± 2.104% to 74.66 ± 1.72% of mupirocin released. At 1 h, 80% of the mupirocin in the scaffold was seen to have diffused out of the scaffold. Release of mupirocin was modulated; an initial burst release was followed by sustained release over 2 h. Electrospun keratin/Co Q10/PVA scaffold containing mupirocin showed excellent antimicrobial activity against all the clinical isolates of 2586, Staphylococcus aureus 2590, 2583, 2587, 2555. All the electrospun scaffolds showed higher cell viability values than the control at 48 and 72 h, with the optimized CoQ10 scaffold concentration being 0.05% w/w. Conclusion: Electrospun nanofibers combining the biocompatibility potential of PVA with the bioactive nature of keratin (0.01% w/w) and CoQ10 (0.5% w/w) and the antibacterial property of mupirocin as a new potential for proper wound care was successfully developed. The cell line studies on this electrospun scaffold (PKCM 3) showed their ability to support the growth of keratinocytes hence the potential of developed scaffolds as a wound dressing. In vivo studies to further investigate the applications of the electrospun keratin/Co Q10/PVA nanofibrous scaffold as a wound dressing is however required.