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Novel nanomaterials have been developed for antimicrobial and wound healing applications. Here, we report the preparation of a polyvinyl alcohol/chitosan (PVA/CS) nanofiber with carboxymethyl chitosan nanoparticles (CMCS-OH30 NPs) encapsulating the antibacterial peptide OH-CATH30 (OH-30). The PVA/CS nanofibers containing OH-30 NPs (NP-30-NFs) obtained via electrospinning could achieve a secondary embedded OH-30. The effect of NP-30-NFs on the release of OH-30 was investigated through high-performance liquid chromatography. The antibacterial activities of NP-30-NFs against Escherichia coli and Staphylococcus aureus were studied by bacterial plate counting. NP-30-NFs containing different concentrations of NPs were applied to mouse skin wounds to determine their effectiveness in promoting wound healing. Results showed that NP-30-NFs exhibited antibacterial properties and promoted skin wound healing.