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Aims: To evaluate the effect of a bacteriocin-activated polythene film on resting and growing populations of Listeria monocytogenes. Methods and Results: The active polythene films were industrially obtained by coating a solution of bacteriocin 32Y from Lactobacillus curvatus upon the surface of the film to be in contact with the packaged material. The behaviour of live Listeria populations was examined in liquid suspensions directly in contact with the bacteriocin-activated film, packed in antimicrobial film, and in a challenge test of storage of frankfurters superficially contaminated by L. monocytogenes and packed in antimicrobial film. In all the experiments, live and dead cells of L. monocytogenes were counted in epifluorescence microscopy after viable staining, which proved to be a suitable method to evaluate the action of bacteriocins on populations of L. monocytogenes. The results showed that the direct contact between active film surface and L. monocytogenes cells is effective for a fast and irreversible inactivation of the population by determining a direct cell disruption. This was confirmed by the results of the challenge test indicating that the antimicrobial package was effective in inhibiting the growth and survival of the pathogen on the surface of frankfurters during storage. Conclusions: The use of the antimicrobial film is encouraged especially for solid food products where the superficial contaminants come immediately in contact with the antimicrobial film. Significance and Impact of the Study: A fast inactivation of the bacterial population, coupled with appropriate conditions of storage, can improve the quality and safety and prolong the shelf-life of the food products packed in antimicrobial films.