Ribosome rescue through the trans-translation pathway is a target for broad-spectrum antibiotic discovery. Broad-spectrum antibiotics can have adverse effects on beneficial bacteria in the human gut microbiome. The effects of trans-translation inhibitors on the microbiome were investigated using strains from two phyla prevalent in the microbiome. Growth inhibitory analysis suggests that these compounds inhibit pathogenic bacterial species, but not beneficial bacteria. These results present potential for novel, broad-spectrum antibiotics that do not harm the microbiome.