Coronopus didymus is a medicinal plant used traditionally as antipyretic, expectorant, blood purifier and for alleviating symptoms of pain, inflammations, malaria, wounds and cancer. The present study was designed with the objectives of revelation of diagnostic compounds responsible for biological activity of this plant. This work is divided into two main parts, part I includes extraction, preliminary phytochemical analysis, isolation, purification, and identification of phenolic constituents by using mass spectrometric technique. Part II comprises bioevaluation of plant for cytotoxic, antioxidant and antimicrobial activities. Total phenolic content of various solvent extracts was determined using the Folin-Ciocalteu assay. Polar and non-polar constituents of C. didymus were identified by ultra performance liquid chromatography-electrospray ionization-mass spectrometric (UPLC-ESI-MS) and gas-chromatography-mass spectrometric analysis (GC-MS), respectively. Major constituents of C. didymus were isolated by using Sephadex LH-20 column chromatography and preparative mass directed HPLC method. The phenolic content was found to be the highest in the ethanolic extract (CDA Et, 47.8 mM GAE). Eighteen phytochemicals were tentatively identified from ethanolic extract by UPLC-ESI-MS analysis. The predominant compounds of aerial parts were flavonoids while roots contained glucosinolate as the major component. Sixty-three volatile phytochemicals were identified from aerial parts and roots of C. didymus by GC-MS analyses. Mass spectrometric profiling of the C. didymus extracts, confirmed that this plant contained a plenty of compounds. The structures of isolated compounds were elucidated by UV-Vis. spectroscopy (with shift reagents), LC-ESI-MS and NMR spectral data as 5,7,4''trihydroxy-3''-methoxyflavone-4''-O-β-D-glucoside (1''), 5,7,4''-trihydroxy-3''methoxyflavone-4''-O-(6''''-acetyl)b -D-glucoside (2'') and 5,7,4''-trihydroxy-3''-methoxy flavone (3''). Compound 1'' was identified for the first time from the genus Coronopus. Extracts, fractions and isolated compounds were subjected to various bioassays in a dose dependent manner to understand their anticancer, antioxidant, antifungal and antibacterial susceptibility. Isolated compounds 1''-3'' showed promising activity against HeLa cells with IC50 values of 43.50, 0.63 and 3.67 µM, respectively. Significant result was also obtained with compound 3'' against LN18 cells with IC50 value of 46.63 µM. In antioxidant activity, compound 3'' was far more effective in scavenging free radicals in the DPPH and ABTS assays with IC50 of 43.8 and 0.08 µg/mL, than the standard trolox, with IC50 values of 97.5 and 21.1 µg/mL, respectively. Compound 3'' depicted largest zone of inhibition (15 mm) against X. campestris, which was even higher than the chemical bactericides viz., bismerthiazole and kasugamycin, with inhibition zone of 11 mm and 14 mm, respectively. Mass spectrometric profiling and the cytotoxic, antioxidant and antimicrobial activity of the phenolic constituents 1''-3'' provide a scientific basis for the ethnopharmacological use of C. didymus for various applications.