In this study, thin, polymeric films consisting of poly(vinyl alcohol) (PVA) and chitosan (Ch) with the addition of poly(hexamethylene guanidine) (PHMG) were successfully prepared. The obtained materials were analyzed to determine their physicochemical and biocidal properties. In order to confirm the structure of PHMG, nuclear magnetic resonance spectroscopy (1H NMR) was applied, while in the case of the obtained films, attenuated total reflectance infrared spectroscopy with Fourier transform (FTIR-ATR) was used. The surface morphology of the polymer films was evaluated based on atomic force microscopy. Furthermore, the mechanical properties, color changes, and thermal stability of the obtained materials were determined. Microbiological tests were performed to evaluate the biocidal properties of the new materials with and without the addition of PHMG. These analyses confirmed the biocidal potential of films modified by PHMG and allowed for comparisons of their physicochemical properties with the properties of native films. In summary, films consisting of PVA and PHMG displayed higher antimicrobial potentials against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria in comparison to PVA:Ch-based films with the addition of PHMG.