AbstractIt is generally accepted that fungi have a number of dormant gene clusters for the synthesis of secondary metabolites, and the activation of these gene clusters can expand the diversity of secondary metabolites in culture. Recent studies have revealed that the mycolic acid-containing bacterium Tsukamurella pulmonis activates dormant gene clusters in the bacterial genus Streptomyces. However, it is not clear whether the mycolic acid-containing bacteria activate dormant gene clusters of fungi. We performed co-culture experiments using marine-derived Aspergillus niger with Mycobacterium smegmatis, a mycolic acid-containing bacteria. The co-cultivation resulted in the production of a pigment by A. niger and increased cytotoxic activity of the extract against human prostate cancer DU145 cells. An analysis of secondary metabolites in the extract of the co-culture broth revealed that the increase in cytotoxic activity was caused by the production of malformin C (1), and that TMC-256A1 (2), desmethylkotanin (3), and aurasperone C (4) were selectively produced under co-culture conditions. In addition, further study suggested that direct interaction between the two microorganisms was necessary for the production of the pigment and the cytotoxic compound malformin C (1) from A. niger. Given the biological activities of malformin C, including cytotoxic activity, our approach for increasing the production of bioactive secondary metabolites has important practical applications and may facilitate structural analyses of novel bioactive compounds.