Brassica tournefortii is an annual herbaceous plant, native to the North Africa and Middle East. It is considered as an excellent medicinal plant due to its richness by antioxidant like isothiocyanates and polyphenols. The present study is the first phytochemical investigation on Brassica tournefortii organs (leaves, stems, and roots) in terms of nutraceutical, chemical composition, and bioactivity. Brassica tournefortii leaves exhibited the highest values of nutraceutical contents. Interestingly, gas chromatograph-y-mass spectrometry (GC-MS) analysis enabled to identify three new isothiocyanates: iberverin nitrile and iberin detected only in roots, and iberin nitrile detected in all organs. HPLC chromatograms displayed different profiles depending on organic solvent and extracted organ. Icariin and 5,7-dihydroxy 4-propylcoumarin showed the highest concentrations with 2.3 and 1.3 mg/g of dr among other molecules identified by high performance liquid chromatography (HPLC). Some phenolic compounds were identified in more than one organ extracts such as phenoxodiol and 4-hydroxy-3-propylbenzoic acid methyl ester. Brassica tournefortii extracts showed a moderate total phenolic contents and anti-15-LOX activity, while they exhibited a good anti-α-glucosidase activity ranging from 40% to 60%. Furthermore, leaves-MeOH and root-dichloromethane (DCM) extracts induced the highest cytotoxicity against MCF-7 cell lines, while roots-cyclohexane (CYHA) extract highlighted the highest inhibition activity against, both, HCT-116 and OVCAR cell lines.