Background: Bacteriocins are antimicrobial peptides with properties making them applicable for food preservation and pathogen control. However, their impact on the gut microbiota remains understudied. In this study we sought to examine the inhibitory spectrum of 75 class II bacteriocins against 49 representative species of the human gut microbiota including 21 Biosafety level 2 organisms. The bacteriocins were cloned and expressed in Escherichia coli and evaluated in vitro, ex vivo and in vivo. Results: In vitro spot assays using Escherichia coli BL21-AI expressing these bacteriocins revealed that 22 bacteriocins inhibited at least one species, with greater efficacy against Gram-positive than Gram-negative species. Two bacteriocins, Actifencin and Bacteroidetocin A, were selected based on their broad spectrum of inhibition for further characterization and applied to murine feces ex vivo. Application of these bacteriocins led to substantial modifications in the composition of the microbial community of murine feces. Yet, these findings could not be replicated in vivo when bacteriocin producing Escherichia coli Nissle strains were dosed to mice. Conclusions: This study evaluate the potential and expands our knowledge of the inhibitory spectrum of class II bacteriocins against a large and representative collection of bacterial isolates from the gut microbiota and underscores that further optimization is needed to use bacteriocins in vivofor modulating microbial community composition.