The seeds of Securigera securidaca (Fabaceae) were used for treatment of diabetes in folk medicine of Iran. In this study the cytotoxicity of different fractions of seeds extract led to purification of potent cytotoxic flavonoids. In the examination of cytotoxicity with brine shrimp lethality test method petroleum ether and chloroform fractions and chloroform (C6) subfraction showed best cytotoxicity with IC50 about 20–22 lg/ml. All fractions and effective subfractions (mortality more than 50 %) were selected for investigation of cytotoxicity measured by MTT assay on three cancer cell lines: colon carcinoma (HT-29), breast ductal carcinoma (T47D), colorectal adenocarcinoma (Caco-2), and normal cell line (NIH/3T3). Results showed cytotoxicity of some chloroform and methanol subfractions against HT-29 and T47D. Three flavonoid glycosides have been isolated from some methanol subfractions. This is the first report for these compounds from seeds extract of S. securidaca. Their structures were determined as orobol-40 -O-glucopyranoside 1, isorhamnetin 3-O-neohesperidoside 2, and isosalipurposide 3. Compounds 1 and 2 showed potent cytotoxicity against abnormal cells in comparison to Methotrexate. All of the fractions and compounds demonstrated no toxicity on NIH/ 3T3 normal cells, means the selective toxicity of plant on cancer cell lines and safety of folkloric usage.