1,2:5,6-Dianhydrogalactitol (DAG) is a bi-functional DNA-targeting agent currently in phase II clinical trial for treatment of temozolomide-resistant glioblastoma (GBM). In the present study, we investigated the cytotoxic activity of DAG alone or in combination with common chemotherapy agents in GBM and prostate cancer (PCa) cells, and determined the impact of DNA repair pathways on DAG-induced cytotoxicity. We found that DAG produced replication-dependent DNA lesions decorated with RPA32, RAD51, and γH2AX foci. DAG-induced cytotoxicity was unaffected by MLH1, MSH2, and DNA-PK expression, but was enhanced by knockdown of BRCA1. Acting in S phase, DAG displayed selective synergy with topoisomerase I (camptothecin and irinotecan) and topoisomerase II (etoposide) poisons in GBM, PCa, and lung cancer cells with no synergy observed for docetaxel. Importantly, DAG combined with irinotecan treatment enhanced tumor responses and prolonged survival of tumor-bearing mice. This work provides mechanistic insight into DAG cytotoxicity in GBM and PCa cells and offers a rational for exploring combination regimens with topoisomerase I/II poisons in future clinical trials.