Three new benzodiazepine alkaloids, circumdatin A (1), B (2), and C (3), were isolated from a terrestrial isolate of the fungus Aspergillus ochraceus. UV-guided separation was used for their purification. The structures of the two zwitterionic benzodiazepines 1 and 2 (differing by a methoxy group in 1 vs. hydrogen in 2) were solved using recently developed 1H-detected INEPT2- and HMBC-INADEQUATE NMR experiments, representing the first naturally occurring zwitterionic benzodiazepines. Compound 3's structure was elucidated via comparative NMR analysis with known benzodiazepines (asperlicin, benzomalvin A) and 13C chemical shift calculations. Hydrolysis and chiral analysis (Marfey's reagent, chiral TLC) revealed the proline moiety in 1 and 2 as L-proline and the alanine moiety in 3 as L-alanine. Chemotaxonomic studies showed that circumdatins A, B, and C are good markers for A. ochraceus, A. ostianus, and A. petrakii. Circumdatin B (2) was tested in the NCI's 60 cancer cell line panel but was not found active.