An earlier study on the marine ascidian Polysyncraton lithostrotum resulted in the isolation of a potent antitumor compound, namenamicin. Because of the remarkable similarity of its enediyne aglycon to that found in the actinomycete-derived calicheamicins and esperamicins, it was suspected that the real producer of namenamicin could be a microbial symbiont. Therefore, a number of actinomycetes were isolated from the inner core of the host ascidian. Among these actinomycetes, a halophilic strain, LL-37I366, was identified as a new species of the genus Micromonospora on the basis of its morphological properties and 16S rDNA sequence, and was named "Micromonospora lomaiVitiensis". The fermentation broth of this organism exhibited potent DNA-damaging activity indicated by the biochemical induction assay (BIA) and was extremely cytotoxic against a panel of cancer cell lines. Using BIA-guided fractionation, two novel dimeric diazobenzofluorene glycosides were isolated and designated lomaiviticins A (1) and B (2). Lomaiviticin A (1) has a molecular formula of C68H80N6O24 and is a symmetric dimer, containing 5,8-dihydroxy-1,4-naphthoquinone substructures, sugar moieties A (N,N-dimethylamino-substituted pyrrolosamine) and B (3-OCH3-substituted sugar), and a dimeric 3,4-dihydroxy-3-ethylhexenone central region with two diazo functional groups. Lomaiviticin B (2) has a molecular formula of C54H56N6O18, is also a symmetric dimer, lacks sugar moieties B, and has a fused furanol central system, with other parts identical to lomaiviticin A (1). Both lomaiviticins A and B are potent DNA-damaging agents with a minimum induction concentration ≤0.1 ng/spot (via BIA). Lomaiviticin A showed cytotoxicity against cancer cell lines with IC50 values ranging from 0.01 to 98 ng/mL and potent antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Enterococcus faecium (MIC 6–25 ng/spot). Although no namenamicin-like compounds were detected in the fermentation broth of LL-37I366, the discovery of the potent antitumor lomaiviticins provides an example of a marine-invertebrate-associated microorganism that is an excellent resource for new bioactive natural products.