Topoisomerase II (Top2) is an essential enzyme for survival of growing cells. This study aimed to screen ethnomedicinal plants used in Thai traditional medicine for Top2 poison activity using a yeast cell-based assay. Mutant yeast strains carrying the top2-1 allele (JN394t2-1), which encodes a temperature-sensitive topoisomerase, were developed to address the mechanism of action of agents with Top2 poison activity. Six out of thirty extracts; Curcuma longa, C. zedoaria, Derris scandens, Grangea maderaspatana, Stephania pierrei and S. suberosa, were found to have Top2 poison activity against the yeast cells. Further phytochemical studies are needed for the isolation and identification of the natural Top2 poison agents found in D. scandens Benth. (Leguminosae). This is the first time that the Top2 poison activities of D. scandens have been studied. Bioassay-guided fractionation based on Top2 poison activity led to the isolation of two isoflavones from the ethyl acetate extract of the stem of D. scandens. The compounds were identified as 5,7,4'-trihydroxy-6,8-diprenylisoflavone and lupalbigenin from spectral evidence. These compounds showed cytotoxicitiy against human epidermoid carcinoma of oral cavity (KB), breast adenocarcinoma (MCF-7) and human small cell lung carcinoma (NCI-H187) cell lines. In DNA Top2 relaxation assay, 5,7,4'-trihydroxy-6,8-diprenylisoflavone and lupalbigenin also showed inhibitory activity with IC₅₀ values of 24.4 and 8.7 µM, respectively. This is the first time that Top2 poison activity of D. scandens has been reported.