Preventing microbial infections and accelerating wound closure are essential in the process of wound healing. In this study, various concentrations of carvacrol (CA) were loaded into polyacrylonitrile/poly-(ethylene oxide) (PAN/PEO) nanofiber membranes to develop potential wound dressing materials via an electrospinning technique. The morphology and structure of the PAN/PEO/CA nanofiber membrane were analyzed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. Subsequently, antimicrobial performance testing showed that the PAN/PEO/CA nanofiber membrane exhibited antimicrobial activity in a concentration-dependent manner. Moreover, SEM and transmission electron microscopy revealed that the number of Staphylococcus aureus decreased significantly and the microstructure of the biofilm was seriously damaged. Next, compared with the control and PAN/PEO groups, the PAN/PEO/5% CA group in a full-thickness skin infection model not only exhibited reduced wound exudate on day 2 after infection but also displayed a greater ability to achieve complete skin regeneration, with faster wound healing. Finally, the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the downregulated differentially expressed genes between PAN/ PEO-and PAN/PEO/5% CA-treated S. aureus were enriched in the two-component system and S. aureus infection. In conclusion, the antimicrobial materials of PAN/PEO/CA inhibited microbial growth and promoted wound healing with potential applications in the clinical management of wounds.