Human DNA topoisomerases have become attractive targets for developing more effective anticancer drugs. In this study, a series of new benzofuro[3,2-b]pyridin-7-ols were designed and synthesized for the first time and screened for their topoisomerase I and II inhibitory and antiproliferative activity. Structure-activity relationships revealed the position of ortho- and para-hydroxyl group at 2-phenyl ring, and meta-hydroxyl group at 4-phenyl ring of benzofuro[3,2-b]pyridin-7-ol are important for potent and selective topo II inhibitory activity. Compound 11 showed the most selective and potent topo II inhibition (100% inhibition at 100 µM) and strongest antiproliferative activity (IC50 = 0.86 µM) than all the positive controls in HeLa cell line.