Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G2 checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G2 checkpoint inhibitors. It inhibited the G2 checkpoint with an IC50 of 8 μM and showed moderate cytotoxicity (IC50 = 25 μM) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC50 = 3 μM) and Chk2 (IC50 = 3.5 μM) but not ataxiatelangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.