As a part of our ongoing collaborative effort to discover the anticancer activity of the phenolics isolated from terrestrial plant sources, the EtOH extract of the aerial parts of the Egyptian medicinal plant Diplotaxis harra (Forsk.) Boiss. was in vitro investigated for its cytotoxicity against HCT116, HepG2 and MCF-7 cell lines, and resulted with IC50=4.65, 12.60 and 17.90 μg/ml, respectively. Doxorubicin (+ve control) showed in vitro cytotoxic activity with IC50=3.64, 4.57 and 2.97 μg/ml, respectively. The phenolic-rich fraction of the EtOH extract was subjected to further fractionation, which led to the isolation of five flavonoids identified as quercetin, quercetin 3-O-β-glucoside, isorhamnetin 7-O-β-glucoside, apigenin 3-O-β-rhamnoside and kaempferol 3-O-β-glucoside, according to its’ spectral data and comparison with the literature. Furthermore, the isolated flavonoids showed in vitro cytotoxicity against HCT116 cell line with IC50=20.1, 24.3, 22.8, 23.4 and 41.9 μg/ml as determined by MTT method.