Methicillin-resistant Staphylococuss aureus (MRSA) is a major nosocomial pathogen that has developed resistance to many antibiotics. New anti-infective drugs to prevent and treat MRSA infection are required. Four assay systems were conducted to screen microbial cultures for new anti-infective compounds active against MRSA. Nosokomycins, new members of the phosphoglycolipid family, were discovered from a culture of Streptomyces cyslabdanicus K04-0144 in an MRSA-silkworm infection assay. The target molecule of nosokomycins was suggested to be the transglycosylase of penicillin binding protein (PBP) involved in MRSA peptidoglycan biosynthesis. Spirohexaline, with a hexacycline structure, was isolated from a fungal culture of Penicillium brasilianum FKI-3368 in an enzyme assay of undecaprenyl pyrophosphate (UPP) synthase, which is needed for the synthesis and transport of GlcNAc–MurNAc–pentapeptides from the cytoplasmic membrane site to the external membrane site for peptidoglycan synthesis. Spirohexaline inhibited MRSA growth by the blockade of UPP synthase activity. Cyslabdan, with a cysteine-carrying labdan skeleton, was also discovered from the nosokomycin-producing actinomycete as a potentiator of imipenem activity against MRSA. The molecular target of cyslabdan was identified as FemA, which is involved in the synthesis of a pentaglycine interpeptide bridge in MRSA peptidoglycan. Citridone A with a unique 6-6/5/5-ring system containing a rare phenyl-R-furopyridone skeleton, originally isolated as a potentiator of antifungal miconazole activity, was found to inhibit MRSA yellow pigment production. These new microbial products will serve as lead compounds for developing new anti-infective drugs for combating MRSA.