A new series of thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyridines containing hydroxyl groups at the ortho, meta, or para position of 2- and 6-phenyl rings attached to the central pyridine were designed and synthesized. They were evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds with hydroxyl moiety either at the meta or para position of 2- or 6-phenyl ring in combination with thienyl or furyl group at 4-position of central pyridine displayed significant topoisomerase II inhibitory activity and cytotoxicity. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the compounds 9-11, 15-17, 19, 21-23, 28, and 41. Among all the synthesized compounds, compound 17 emerged as the most promising topoisomerase II inhibitor with significant cytotoxicity.