On the basis of previous reports on the importance of thienyl, furyl or phenol group substitution on 5H-indeno[1,2-b]pyridine skeleton, a new series of rigid 2-aryl-4-(4'-hydroxyphenyl)-5H-indeno[1,2-b]pyridine derivatives were systematically designed and synthesized. Topoisomerase inhibitory activity and antiproliferative activity of all the synthesized compounds were determined using human colorectal (HCT15), breast (T47D), prostate (DU145) and cervix (HeLa) cancer cells. Compounds 9, 10, 12, 13, 15, 16, 18 and 19 with thienyl or furyl moiety at the 2-position and hydroxyl group at the meta or para positions of 4-phenyl ring displayed strong to moderate topoisomerase IIα (topo IIα) inhibitory activity and significant antiproliferative activity. The evaluation of compound 16 to determine its mechanism of action was performed with topo IIα-DNA cleavable complex, topo IIα-mediated ATPase assay, DNA unwinding and in vitro and ex vivo topo IIα relaxation assay. Compound 16 functioned as a DNA non-intercalative topo IIα catalytic inhibitor with better potency than etoposide in T47D breast cancer cells. Molecular docking study revealed that compound 16 cannot intercalate into regularly stacked base-pairs of DNA duplex but can interact or intercalate to topo IIα-bound DNA.