Staphylococcus aureus is a leading cause of infection in the United States, and due to the rapid development of resistance, new antibiotics are constantly needed. trans-Translation is a particularly promising antibiotic target because it is conserved in many bacterial species, is critical for bacterial survival, and is unique among prokaryotes. We have investigated the potential of KKL-40, a small-molecule inhibitor of trans-translation, and find that it inhibits both methicillin-susceptible and methicillin-resistant strains of S. aureus. KKL-40 is also effective against Gram-positive pathogens, including a vancomycin-resistant strain of Enterococcus faecalis, Bacillus subtilis, and Streptococcus pyogenes, although its performance with Gram-negative pathogens is mixed. KKL-40 synergistically interacts with the human antimicrobial peptide LL-37, a member of the cathelicidin family, to inhibit S. aureus but not other antibiotics tested, including daptomycin, kanamycin, or erythromycin. KKL-40 is not cytotoxic to HeLa cells at concentrations that are 100-fold higher than the effective MIC. We also find that S. aureus develops minimal resistance to KKL-40 even after multiday passage at sublethal concentrations. Therefore, trans-translation inhibitors could be a particularly promising drug target against S. aureus, not only because of their ability to inhibit bacterial growth but also because of their potential to simultaneously render S. aureus more susceptible to host antimicrobial peptides.