Developing efficient hydrogel‐based wound dressings has gained increasing attention in improved wound healing capabilities. In this study, the biocomposite hydrogel films consisting of polyvinyl alcohol (PVA), chitosan (CS), and zinc oxide nanoparticles (ZnO NPs) were prepared using the solvent casting method. The biocomposite hydrogel films were also loaded (5% w/w) with Amphotericin B (AmB) as a model drug to develop a new suitable material with potential application as high‐performance wound dressing with controlled drug release. The chemical nature, thermal properties, and morphology of the PVA/CS and PVA/CS‐ZnO biocomposite hydrogels were analyzed and compared by FTIR, TGA, and SEM–EDX analysis. The effect of ZnO NPs on swelling behavior in the PVA/CS films was studied. PVA/CS‐ZnO biocomposite hydrogels presented a lesser degree of swelling compared to ZnO‐free hydrogels. The AmB‐loaded hydrogel films exhibited a sustained release rate during 72 h in PBS at pH 7.4. Biocompatibility of all hydrogel types (with or without ZnO and AmB) using L929 and HaCaT cell lines was evaluated with high cell viability examined by MTT assay. The antimicrobial activity of the films was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans using a modified AATCC 100 method, whereby the R value represents the log reduction in the number of surviving colonies. The mean R values for drug‐free PVA/CS‐ZnO hydrogel films were 6.81 for E. coli, 6.03 for S. aureus, and 6.82 for C. albicans. The addition of AmB further enhanced the antimicrobial activity, increasing the R values for E. coli to 7.16, for S. aureus to 6.38, and for C. albicans to 7.31, demonstrating a significant synergistic effect with maximum antimicrobial efficacy. The results showed that the AmB‐loaded biocomposite hydrogel films (PVA/CS‐ZnO‐AmB) can be used as a new wound dressing material.