This work is focused on novel methodology of poly(vinyl alcohol) crosslinking by non-toxic dicarboxylic acid, glutaric acid. The cross-linked system was used as a matrix for immobilization of bacteriocin nisin. Effect of the crosslinking degree on physico-chemical, morphology, mechanical, and thermal properties of poly(vinyl alcohol) films were investigated by using swelling test, Fourier transform infrared spectroscopy, scanning electron microscopy, stress-strain analysis, differential scanning calorimetry, and thermogravimetry. Release profile of the nisin from the cross-linked poly(vinyl alcohol) was studied by high performance liquid chromatography. Antibacterial activity of the prepared systems was tested by agar diffusion test and dilution and spread plate technique. Results showed suitability of glutaric acid as effective crosslinking agent of poly(vinyl alcohol) that acts synergistically with bacteriocin nisin against the tested Gram-positive and Gram-negative bacterial strains. V C 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43674.