Nogalamycin is an anthracycline anti-cancer agent that intercalates into the DNA double helix. The binding is facilitated by two carbohydrate units, L-nogalose and L-nogalamine, that interact with the minor and major grooves of DNA, respectively. However, recent investigations have shown that nogalamycin biosynthesis proceeds through the attachment of l-rhodosamine (2′′-deoxy-4′′-epi-L-nogalamine) to the aglycone. Herein, we demonstrate that the Rieske enzyme SnoT catalyzes 2′′-hydroxylation of L-rhodosamine as an initial post-glycosylation step. Furthermore, we establish that the reaction order continues with 2–5′′ carbocyclization and 4′′ epimerization by the non-heme iron and 2-oxoglutarate-dependent enzymes SnoK and SnoN, respectively. These late-stage tailoring steps are important for the bioactivity of nogalamycin due to involvement of the 2′′- and 4′′-hydroxy groups of ᴸ-nogalamine in hydrogen bonding interactions with DNA.