Antimicrobial agents can be used to give antimicrobial properties to polymeric materials used to envelope foods for packaging purposes. In this study, we exploited an atomic force microscopy (AFM) analysis for the characterization of plastic films activated with antimicrobial agents. The aim was to acquire information on the distribution of the antimicrobials on plastic materials with the ultimate scope of understanding the mechanisms of interaction between antimicrobials and materials to be used for food packaging. Four polyethylene films differing in linear, EVA, and erucamide content were activated by 3 different bacteriocins as antimicrobials, namely, nisin and bacteriocins Bac162W from Lactobacillus curvatus and BacAM09 from Lactobacillus plantarum. The spectrum of activity of the bacteriocins was assayed and shown to include several strains of Listeria monocytogenes. The plastic films were activated by a previously developed coating procedure and the surfaces of the active films were examined by AFM. In addition, roughness parameters related to the single surfaces were investigated by an appropriate software. Significant differences were found between the bacteriocin activated and control (nonactivated) films and the activated surfaces showed lower values of average roughness and surface area ratio. It was not always possible to obtain a homogeneous distribution of the bacteriocin preparation following the coating procedure. This result was dependent on the bacteriocin used and its distribution on the different plastic films. Overall, the bacteriocin Bac162W showed the most homogeneous distribution while surfaces treated with nisin, showing a sort of microtexturing, always gave the highest roughness values. Although the issue needs further investigation, the connection between AFM imaging, roughness, and antimicrobial distribution on active packaging showed the potential to improve the understanding of the interactions between plastic films and antimicrobial preparations that can be important for the innovation in food packaging and science.