An extracellular matrix-mimicking, biodegradable tissue-engineered skin substitute with improved antibacterial, antibiofilm, and wound healing capabilities is essential in skin tissue regeneration applications. The purpose of this study was to develop a novel biodegradable composite nanofibrous poly(ε-caprolactone) (PCL)/decellularized extracellular matrix (dECM) scaffolds loaded with usnic acid (UA); (PEU), where UA is employed as an antibacterial agent as well as a wound-healing accelerator. The architecture and fiber structure of the scaffolds were examined using scanning electron microscopy, and the results revealed that the average diameters decreased as the dECM content increased. The chemical composition, changes in the crystalline structure, homogeneity, and thermal stability of the nanofiber scaffolds with different material compositions were determined using Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis, respectively. The composite nanofibrous scaffolds exhibited strong antibacterial activity against various bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Cutibactrium acnes, and fungal pathogens (such as Candida albicans). Additionally, the composite nanofibrous scaffolds exhibited biofilm inhibition properties against Klebsiella pneumoniae and Pseudomonas aeruginosa. An evaluation of the appearance of in vivo full-thickness excisional wounds treated with the composite nanofiber scaffolds, as well as a histological analysis of the wounds 21 days after surgery, revealed that treatment with nanofibrous PEU scaffolds enhanced wound healing. This study reveals that the proposed composite nanofibrous PEU scaffold has substantial potential for treating infectious full-thickness wounds.