Six flavonoid glycosides ( 1 – 3 , 6 – 8 ), one lignan ( 4 ), and one galloyl glycoside ( 5 ) were isolated based on the phytochemical study for the MeOH extract of Polygonum aviculare L. herb. The eight isolated compounds' structures were determined by comparing their physicochemical and spectral data with published ones. Compounds 5 and 6 were first isolated from this plant, and all the compounds were evaluated for DNA topoisomerase inhibitory activities and cytotoxicities. Among the purified compounds, 5 showed stronger inhibitory activity against topoisomerase I with an IC 50 value of 12.0 μM, while the value of positive control camptothecin (CPT) was 17.0 μM. For compounds 1 , 3 , and 5 – 8 , the value ranged from 0.054 to 17.0 µM, suggesting more potent inhibitory activity against topoisomerase II, and that of etoposide (VP-16) was 28.0 µM. Particularly, compounds 6 and 8 (IC 50 = 0.054 and 0.077 µM, respectively) inhibited Topo II about 500 times stronger than VP-16, making them safer agents of DNA topoisomerase. Furthermore, all compounds demonstrated no cytotoxic activity against four cancer cell lines. This study is the first to report the inhibition of DNA topoisomerases of compounds from P. aviculare , suggesting that these compounds may be potential candidates for treating cancer.