In this work, gelatin/transglutaminase and gelatin/chitosan/transglutaminase films were obtained by casting onto a plastic substrate. The mechanical and sorption properties of the films were studied depending on the transglutaminase content. Treatment of gelatin with transglutaminase led to conformational changes in its secondary structure. Deconvolution of the IR spectra of gelatin/transglutaminase films allowed us to determine an increase in the proportion of β-sheets and a decrease in the proportion of random coils, which had a positive effect on the mechanical properties of these films. Cross-links formed by introducing transglutaminase led to structural changes in gelatin and the formation of a dense spatial polymer network, due to which the solubility and equilibrium degree of swelling of gelatin/transglutaminase films were reduced. The introduction of a small amount of transglutaminase significantly reduced the rate of water sorption over the entire time interval and led to an increase in tensile strength for films of the gelatin/chitosan/transglutaminase composition. Excessive cross-linking of gelatin/chitosan/transglutaminase films resulted in structural changes that negatively affected the mechanical properties.