Two glycosylated naphthacemycins (naphthacemycins D1 and D2) were identified in Streptomyces sp. N12W1565. These two compounds not only showed antimicrobial potential against bacteria but also exhibited more aqueous solubility than naphthacemycins. Furthermore, the whole genome of Streptomyces sp. N12W1565 has been sequenced, the natY gene, located outside the biosynthetic gene cluster encoding a D-glucose glycosyltransferase, was identified to mediate glycosylation in the phenolic hydroxyl of the naphthacemycin core scaffold. Glycosyltransferase was elucidated in vitro by using a homologous enzyme, which showed potential as a biocatalyst.