Abstract <p>Cancer is a predominant contributor to global morbidity and mortality, affecting populations worldwide. Marine <i>Micromonospora</i> species have been identified as significant sources of anticancer compounds. This work aimed to perform a polyphasic approach of isolated strain and conduct comparative metabolomic and genomic analyses to identify compounds with anticancer activity. The study utilized a polyphasic approach on isolated strains for anticancer compound identification. Taxonomic analysis of strain 2MTK254 revealed unique pigment and fatty acid patterns, designating it as a novel <i>Micromonospora carbonacea</i> subsp. <i>caeruleus</i>. Its crude extract displayed significant anti-colorectal activity (66.03% inhibition). Molecular network analysis classified metabolites into eight classes, highlighting a polycyclic tetramate macrolactams (PTMs) compound (P1, C₂₉H₃₈N₂O₄) with 99.31% inhibitory activity against HCT-116 cell lines (IC₅₀ at 0.125&#xa0;µM). Genome analysis identified 32 biosynthetic gene clusters (BGCs), including unique PTMs BGCs (83% similarity) linked to the P1 compound. Thus, <i>M. carbonacea</i> subsp. <i>caeruleus</i> 2MTK254 holds promise as a source of novel PTMs with anti-colorectal cancer potential.</p> Graphical Abstract <p></p> Key points <p>• <i>A novel strain of Micromonospora carbonacea subsp. caeruleus 2MTK254 was isolated in Thailand</i></p> <p>• <i>A new polycyclic tetramate macrolactam (PTM) with anticancer activity was identified in 2MTK254</i></p> <p>• <i>The genome of 2MTK254 has unique secondary metabolite gene clusters</i></p>