The marine environment has become an attractive new source of chemical diversity for drug discovery, as new bioactive substances are isolated from marine organisms, including phytoplankton, algae, sponges, tunicates and mollusks. Our group has reported the isolation of azaspiracid-2 and JBIR-44 from marine sponges. Marine microorganisms, especially fungi, are an untapped resource of novel bioactive substances such as diketopiperazine alkaloids, trichodermatides and carbonarones. We have also reported the discovery of new sesquiterpenes, JBIR-27 and JBIR-28; a new aspochracin derivative, JBIR-15; new glycosyl benzenediols, JBIR-37 and JBIR-38; and a new sorbicillinoid, JBIR-59 from marine-derived fungi. The Calcarea class of sponges has been recognized as a reliable source of richly bioactive metabolites possessing a 2-aminoimidazole skeleton. However, the isolation of fungi from calcareous marine sponges has not been reported. Therefore, we attempted to isolate marine fungi from an unidentified calcareous marine sponge and then searched for secondary metabolites in the cultures of the isolated strains. In this study, we report the isolation of Aspergillus sp. fS14 from this marine sponge, and the fermentation, isolation and structure elucidation of two new compounds, designated JBIR-74 (1) and JBIR-75 (2) (Figure 1a).