The aim of the present study is to determine the potent biological activities and carry out isolation studies on Barbarea integrifolia. The antioxidant capacity of the species was evaluated by total phenolic content, FRAP, CUPRAC, and DPPH radical scavenging activity. Anticancer activity studies were performed by MTT assay in MDA-MB-231, MCF-7, Hep3B, PC-3, A549, HCT116, L-929 cell lines. It was observed that the remaining aqueous fraction has higher total phenolic content while higher activity in the CUPRAC and FRAP assays was displayed for the methanolic extract and chloroform fraction. The extracts showed anticancer activity as compared with vincristine. It was observed that chloroform fraction has the highest anticancer activity on MCF-7 cell line, while ethyl acetate fraction has the highest anticancer activity on Hep-3B and A549 cell lines. Methanolic extract has the highest anticancer activity on HCT116 and MDA-MB-23 cell lines. The isolation studies have been performed using several chromatographic methods. The chemical structures of compounds have been identified by means of H-1 NMR, C-13 NMR, 2D-NMR, and MS. Five major compounds, one steroid (beta-Sitosterol), one phenolic acid (Rosmarinic acid), one flavonol heteroside (kaempferol 7-O-alpha-L-rhamnoside-3-O-beta-D-(2-O-beta-D-glucosyl)-beta-D-glucoside), and two glucosinolates (Gluconasturtiin, Gluconasturtiin choline salt) have been isolated.