•The elasticity of the film was significantly improved by the incorporation of PVA.•WVP and OP were dependent on CS/PVA ratio.•Initial diffusion of Nisin could be well described by the Fickian equation.•pH, ionic strength, film composition and temperature affected Nisin diffusion.•Diffusion of Nisin from film was spontaneous and endothermic. The aim of this study was to evaluate the kinetics and functional effectiveness of Nisin loaded chitosan/poly(vinyl alcohol) (Nisin–CS/PVA) as an antibacterial packaging film. The films were prepared by coating method and Staphylococcus aureus (S. aureus, ATCC6538) was used as test bacterium. The intermolecular hydrogen bonds between CS and PVA molecules were confirmed. The elasticity of films was significantly improved by the incorporation of PVA, and the film could also bear a relative high tensile strength at 26.7MPa for CS/PVA=1/1. As CS/PVA ratio decreased, the water vapor permeability (WVP) decreased and reached its minimum value 0.983×10−10gm−1s−1 at CS/PVA=1/1, meanwhile, oxygen permeability (OP) increased but still lower than 0.91cm3μmm−2d−1kPa−1 for CS/PVA=1/1 as the CS/PVA ratio was above 1:1. The initial diffusion of nisin (Mt/M∞<2/3) from CS/PVA film could be well described by the Fickian diffusion equation. Owing to the positively charged nisin at pH below isoelectric point (pI, 8.8) and its increasing dissolubility in water as the pH reduced, the diffusion of nisin from the films strongly depended on pH and ionic strength besides CS/PVA ratio and temperature. Moreover, the thermodynamic parameters suggested the spontaneous and endothermic diffusion of nisin from the films. The resulting data can provide some valuable information for the design of film in structure and ingredient.