The everninomicins are complex oligosaccharides with broad antimicrobial activity produced by Micromonospora carbonacea. However, toxicity issues and an inability to easily access analogs stifled their previous development. Recent crystal and cryo-EM structures of everninomicin A bound to the bacterial ribosome provide a detailed map of the vital interactions responsible for the everninomicins’ activity. The structures reveal crucial interactions between the 50S subunit and the aromatic dichloroisoeverninic acid (DCE) moiety of everninomicin. All natural everninomicins contain at least one iterative type I polyketide synthase (iPKS)-derived DCE moiety. We propose to access everninomicins with derivatized aromatic moieties via the biosynthetic machinery. In order to gain a better understanding of DCE biosynthesis, we deleted the four putatively associated genes: an iPKS, an O-methyltransferase (O-MT), a flavin-dependent halogenase (FDH), and a trans acyltransferase (AT). Functional analysis of these genes confirmed their assignment and provided seven novel everninomicin metabolites. These results demonstrate that the iPKS EvdD3 is responsible for the biosynthesis of the DCE core scaffold, orsellinic acid. Orsellinic acid is transferred to the terminal D-olivose sugar residue by AT EvdD1 to be subsequently tailored by O-MT EvdM5 and FDH EvdD2 to yield DCE. This work also revealed that transfer of the evernitrose sugar to D-olivose requires the presence of the nearly complete DCE ring. Current work is focused on in vitro analysis of the DCE-associated enzymes to evaluate incorporation of non-natural substrates. This work will further elucidate the biosynthesis of the everninomicins and provide novel analogs to revitalize this potent class of antibiotics.