Two new flavonol glycosides, isorhamnetin 3-O-β-glucopyranoside-4'-O-β-xylopyranoside (1) and kaempferol 3-O-β-glucopyranoside -4'-O-β-xylopyranoside (2), were isolated from the defatted aqueous methanol extract of the whole plant Diplotaxis harra along with 12 known flavonols (3-14). They were characterised by chemical and spectral methods. The 70% aqueous methanol, chloroform and defatted aqueous methanol plant extracts exhibited significant antioxidant effects (nitroblue tetrazolium reduction method). Their cytotoxic activity was carried out against 11 tumour cell lines (sulphorhodamine B assay). The three extracts expressed the greatest antiproliferative activity against colon 38, P388 and MKN-28 with GI50 (0.45, 0.4, 0.07 μg/mL) and against P388 [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay] with IC50 (0.26, 0.24, 0.25 μg/mL), respectively. The chloroform extract showed the highest activity as eukaryotic DNA topoisomerase II inhibitors of P388 with IC50 0.24 μg/mL. Antiviral screening of the extracts and the pure compounds against foot-and-mouth disease virus types A and O revealed a prominent inhibition of its cytopathic effect.