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 the namenamicin could be a microbial symbiont. Therefore, a number of actinomycetes were isolated from the inner core of the host ascidian to answer the question of compound origin. 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). In this paper, the production, isolation, structural elucidation, and biological activity of the new antibiotics are reported. The molecular formula of lomaiviticin A (1) was determined to be C68H80N6O24, and it was a symmetric dimer containing two units of 5,8-dihydroxy-1,4-naphthoquinone, two amino sugar moieties A (N,N-dimethylpyrrolosamine) and B (R-oleandrose), and a dimeric 3,4-dihydroxy-3-ethylhexenone center. The molecular formula of lomaiviticin B (2) was C54H56N6O18, also a symmetric dimer, lacking sugar moieties B and with a fused furanol center instead of the hexenone. Both compounds contain dimeric diazobenzofluorene structures. Lomaiviticins A and B were demonstrated to be potent DNA-damaging agents by BIA, both with a minimum induction concentration ≤0.1 ng/spot. The more abundant lomaiviticin A was also tested against a number of cancer cell lines and showed cytotoxicity with IC50 values ranging from 0.01 to 98 ng/mL and cleaved double-stranded DNA under reducing conditions. Lomaiviticins A and B were also potent antibiotics against Gram-positive bacteria, Staphylococcus aureus and Enterococcus faecium (MIC's, 6-25 ng/spot) in a plate assay. In summary, we have isolated two potent antitumor antibiotics, lomaiviticins A and B, with dimeric diazobenzofluorene glycoside structures, from the cultural broth of "Micromonospora lomaiVitiensis". Although no namenamicin-like compounds were detected from the fermentation broth of LL-37I366, several other actinomycete strains, also isolated from Polysyncraton lithostrotum, exhibited DNA-damaging activity, and their active components will be examined. The results described in this paper did not solve the mystery of the namenamicin origin; however, 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.