Through precursor-directed biosynthesis, feeding halogenated (F-, Cl-, Br-, I-) or methoxy-substituted 4-methyl-3-hydroxyanthranilic acid (4-MHA) analogues to the acnGHLM-deleted mutant strain of Streptomyces costaricanus SCSIO ZS0073 led to the production of ten new actinomycin analogues (4-13). Several of the actinomycin congeners displayed impressive antimicrobial activities, with MIC values spanning 0.06-64 μg/mL to clinically derived antibiotic resistant pathogens, including Staphylococcus aureus, Enterococcus faecium, and Candida albicans, with low cytotoxicity.