Globomycin, a signal peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. One of the analogues showed a more potent activity against Gram-negative bacteria than globomycin and also exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).