In the present study, hydrogel films composed of chitosan-poly(vinyl alcohol)-gelatin-thyme honey were successfully prepared by casting method, and their anti-bacterial properties were modeled and optimized. Antibacterial properties of the prepared films were analyzed by applying agar diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were tested as Gram-positive and Gram-negative bacteria, respectively. In order to obtain the composition of the film with maximum inhibition zone against both above-mentioned bacterial strains, the experiments were designed using response surface methodology based on five-level central composite design with four parameters, including concentrations of chitosan, poly(vinyl alcohol), gelatin, and honey. The results indicated that the prepared samples had good antibacterial activities against these two studied bacteria strains. Response surface method is conducted to develop mathematical models for process responses. Variance analysis on the experimental data shows that inhibition zone can be predicted effectively with quadratic models. In addition, swelling properties and rate of water vapor transmission of the prepared hydrogel films were studied. Due to the successful results, this hydrogel film has an excellent potential to be explored further as a wound healing material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2544-2553, 2016.