During our continued search for bioactive metabolites from pathogenic microorganisms, we isolated a novel tricyclic diterpenoid antibiotic, brasilicardin A, from Nocardia brasiliensis. This study reports its in vitro and in vivo antitumor activity. In vitro, brasilicardin A exhibited cytotoxicity against various cell lines with IC50 values ranging from 0.078 to 100 μg/ml, showing high susceptibility in P388 and adriamycin-resistant P388/ADR cells. In vivo, it significantly prolonged the mean survival time (MST) of P388 and P388/ADR tumor-bearing mice, with T/C values up to 165% for P388/ADR, and showed no obvious body weight loss. Additionally, brasilicardin A did not reverse P-glycoprotein-mediated multidrug resistance in P388/ADR cells.