Diet is an important factor influencing the composition and function of the gut microbiome, but the effect of antimicrobial agents present within foods is currently not understood. In this study, we investigated the effect of the food-grade cationic antimicrobial ε-polylysine on the gut microbiome structure and predicted metagenomic function in a mouse model. The relative abundances of predominant phyla and genera, as well as the overall community structure, were perturbed in response to the incorporation of dietary ε-polylysine. Unexpectedly, this modification to the gut microbiome was experienced transiently and resolved to the initial basal composition at the final sampling point. In addition, a differential non-random assembly was observed in the microbiomes characterized from male and female co-housed animals, although their perturbation trajectories in response to diet remain consistent. In conclusion, antimicrobial ε-polylysine incorporated into food systems transiently alters gut microbial communities in mice, as well as their predicted function. This indicates a dynamic but resilient microbiome that adapts to microbial-active dietary components.