In the course of our investigations of marine and aquatic fungi as sources of novel biologically active secondary metabolites, we examined an isolate of Stachybotrys sp. (CS-710-1) collected from brackish water in Florida. This isolate differs significantly from previously known members of the genus Stachybotrys and has been established as a representative of a new species. Cultures of this new species exhibited activity in competitive assays against other fungi, and efforts were undertaken to isolate the compound(s) responsible for these effects. Through these studies, we have encountered two new antifungal metabolites which we have named stachybotrins A and B. Stachybotrins A and B (1 and 2), two new aromatic alkaloids with antibacterial and antifungal activity, have been isolated from an aquatic isolate of a new species of the genus Stachybotrys (CS-710-1). They were obtained from ethyl acetate extracts of liquid cultures by repeated preparative TLC on silica gel. The structures were determined primarily by analysis of HMBC, HMQC, COSY, and NOESY experiments. Stachybotrins A and B each showed activity in disk assays against Bacillus subtilis (ATCC 6051) at 10 μg/disk (8- and 10-mm zones, respectively). They also inhibited the radial growth of the filamentous fungi Ascobolus furfuraceus (NRRL 6460) and Sorduria fimicola (NRRL 6459) by 50% in centerpoint inoculation assays at 20 and 10 μg/disk, respectively.