An ideal wound dressing should possess high mechanical properties, biocompatibility, and antibacterial and anti-inflammatory properties while being simple and affordable. So, a dual-layer hydrogel-nanofiber wound dressing was fabricated in this study. The hydrogel as the inner layer was prepared using sodium alginate and guar gum, while the nanofiber layer was made of polyvinyl alcohol and chitosan enriched with Ciprofloxacin. Besides the biological and morphological characteristics, the films were evaluated for mechanical properties and swelling behavior and in-vivo experiments were conducted to investigate their wound healing ability. Compared to single-layer hydrogel, the bi-layer hydrogel-nanofiber exhibited excellent mechanical properties, a proportional swelling rate, and water vapor transmission rate, slow degradation towards tissue regeneration, and anti-inflammatory and antimicrobial properties. Tensile strength and elongation at break improved from 0.163 ± 0.55 MPa and 16.869 ± 0.48 % to 1.674 ± 0.41 MPa and 34.062 ± 0.33 %, respectively (p <
0.05). The drug release profile showed an initial burst of 35 ± 1 % within the first hour, followed by controlled release over 24 h. Hemolysis rates were below 2 %, with 1.13 ± 0.03 % for hydrogel-nanofibers, demonstrating excellent blood compatibility. Bi-layer hydrogel-nanofiber significantly enhanced wound healing in rats, resulting in minimal wound surface and denser collagen deposits. Reductions in IL-1β and IL-6 (p <
0.05), confirmed the films' efficacy in wound healing and inflammation mitigation.