Through precursor-directed biosynthesis, feeding halogenated (F-, Cl-, Br-, I-) or methoxy-substituted 4-methyl-3-hydroxyanthranilic acid (4-MHA) analogues to the <i>acnGHLM</i>-deleted mutant strain of <i>Streptomyces costaricanus</i> SCSIO ZS0073 led to the production of ten new actinomycin analogues (<b>4</b>-<b>13</b>). 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 <i>Staphylococcus aureus</i>, <i>Enterococcus faecium</i>, and <i>Candida albicans</i>, with low cytotoxicity.