Homologous recombination repair (HRR), a crucial approach in DNA damage repair, is an attractive target in cancer therapy and drug design. The Bloom syndrome protein (BLM) is a 3'-5' DNA helicase that performs an important role in HRR regulation. However, limited studies about BLM inhibitors and their biological effects have been reported. Here, we identified a class of isaindigotone derivatives as novel BLM inhibitors by synthesis, screening, and evaluating. Among them, compound 29 was found as an effective BLM inhibitor with a high binding affinity and good inhibitory effect on BLM. Cellular evaluation indicated that 29 effectively disrupted the recruitment of BLM at DNA double-strand break sites, promoted an accumulation of RAD51, and regulated the HRR process. Meanwhile, 29 significantly induced DNA damage responses, as well as apoptosis and proliferation arrest in cancer cells. Our finding provides a potential anticancer strategy based on interfering with BLM via small molecules.