AbstractIn this study, diffusional and antimicrobial properties of the active‐multilayer plastic films (LDPE/polyamide/LDPE‐2% antimicrobial agent) were assessed. To understand the diffusion of antimicrobials from the active plastic films containing nisin, potassium sorbate, silver substituted zeolite, or chitosan, the active‐multilayer plastic films has been put in different food simulating solutions. The food simulating solutions had various pH (3.0–7.0) and water activity (0.65–0.95) levels and were incubated at various storage temperatures (5–25°C). The release experiments for the combinations of the factors were set according to three factors face‐centered central composite design (CCF) of the response surface methodology (RSM). The diffusion rate constant (D) was calculated using Fick's law approach. The D of nisin and silver substituted zeolite was affected by pH or temperature, while aw is not a significant factor. However, Dps was found to be the highest and depended on all factors (4.45–10.04 × 10−14 cm2/s). Furthermore, no chitosan release was detected from the film matrix. The antimicrobial effectiveness of the films against microorganisms that were Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger varied according to the film type, which was verified by a challenge test on the kaşar cheese surface. Moreover, nisin and silver substituted zeolite incorporated films showed great potential to inhibit bacteria, yeast, and molds. All active films, which were studied, had featured tasks for being active food packaging material and diffused in allowance limits.