Antibacterial agent 256 (Compound C09) is an inhibitor for type I signal peptidase ( SPase I ). Antibacterial agent 256 inhibits gram-positive bacteria, that inhibits S. aureus ATCC 29213, E. faecium QF31, E. faecalis SF23-1 and S. suis P1/7, with MIC of 1-16 μg/mL. Antibacterial agent 256 exhibits cytotoxicity in cancer cell HEp-2 and Caco-2 with CC 50 of 14.65 μg/mL and 21.93 μg/mL. Antibacterial agent 256 exhibits a hemolytic activity on mouse RBCs, with an HC 50 of 13.29 μg/mL. Antibacterial agent 256 ameliorates the MRSA skin infection in mouse model.