Raphanus raphanistrum L. (Family: Brassicaceae) is an important crop used worldwide for its medicinal and therapeutic properties as well as its high nutritional value. The present work aims to investigate the flavonoid constituents and the cytotoxicity for R. raphanistrum extract. Ten flavonoid compounds were isolated by chromatographic techniques They were identified as kaempferol (1), kaempferol 3-O-α-arabinopyranoside (2), kaempferol 3-O-α- rhamnopyranoside (3), kaempferol 3-O-b -glucopyranoside-7-O-α-rhamnopyranoside (4), kaempferol 3-O-α-rhamnopyranoside-7-O-b -glucopyranoside (5), kaempferol 3,7-di-O-α-rhamnopyranoside (6), kaempferol 3-O-(2¢¢-b -glucopyranosyl)-α-rhamnopyranoside-7-O-α-rhamnopyranoside (7), quercetin 3-O-α- rhamnopyranoside-7-O-α-arabinopyranoside (8), quercetin 3-O-b -glucopyranoside-7-O-α-rhamnopyranoside (9), quercetin 3-O-α-rhamnopyranoside-7-O-b-glucopyranoside (10). Except for compounds 4 and 5, all flavonoids were firstly isolated from the plant. The LC-ESI-MS technique was also performed and revealed the tentative identification of kaempferol and quercetin nuclei with high grade of glycosylation for the first time. Furthermore, moderate cytotoxic activity, for R. raphanistrum extract, against HEPG2 and MCF7 at 100 mg/mL were assisted with cell viability 62.4% and 57.9%, respectively