|Abstract||Staphylococcus aureus quickly develops resistance to antibiotics and poses a significant health threat to humans. New antibiotic targets are needed for the development of new antibiotics. Trans-translation has important roles in maintaining bacterial viability. Small molecules, KKL-35 and KKL-40, were recently identified as specific inhibitors of trans-translation. We have investigated the roles of trans-translation on S. aureus viability and the potential of KKL-35 and KKL-40 as antibiotics. We find that KKLs show bactericidal activity against multiple S. aureus strains at relatively low concentration. We also find that sub-lethal doses of KKLs make S. aureus more susceptible to antimicrobials. Neither KKL-35 nor KKL-40 are cytotoxic to human HeLa cells. Unfortunately, KKL-40 is inactivated by human serum. Therefore, new inhibitors will need to be identified in future studies. Notably, the development of resistance by S.aureus against KKLs remains at a low level. Therefore trans-translation is a promising target for antibiotic development.