Bioresponsive nanodrug delivery systems have excellent potential in tissue engineering applications. Poly-anionic and poly-cationic biopolymers have provided a superior platform for designing pH-sensitive drug delivery systems. In this regard, hyaluronic acid–chitosan–polyvinyl alcohol complex nanofibers with high quality and reproducibility were produced by optimizing the solution preparation process. In addition, the synthesized composite nanofiber, with 66.82 kN/mm toughness, 200% swelling ratio, and 60% porosity, exhibited excellent properties to meet the requirements of the ideal wound dressing. Green cross-linking with citric acid prevented the destruction of the nanofiber even after prolonged immersion in biological solutions. ζ potential studies demonstrated that the synthesized nanofiber has a negative surface charge (∼−30) at physiological pH. The pK a of the temporin-Ra peptide is about 10, and as a result the peptide molecules have a net positive charge in physiological conditions. Therefore, peptide molecules immobilized on the synthesized scaffold based on surface adsorption. In vivo evaluation has proven that the wound bed has an alkaline environment, facilitating peptide release from the nanofiber scaffold. Electrospun nanofibers can imitate the architecture of the extracellular matrix for accelerating wound healing. In vitro investigation showed better adhesion, proliferation, migration, and fibroblast cell growth on peptide-loaded nanofiber samples than other groups. In vivo studies on full-thickness wounds in the mouse model indicated that the designed nanofiber was gradually absorbed without causing dryness or infection. On day 6, the peptide-loaded nanofiber revealed 60% wound closure compared to the control group (17%). In addition, based on histological studies, the composite nanofiber demonstrated excellent tissue repair ability, hence these active nanofiber mats can be a good alternative to existing wound dressings. Gene expression studies show that the antimicrobial peptide promotes the inflammatory phase of wound healing in a shorter time frame by accelerating the tumor necrosis factor-α cytokine response.