The purpose of our study was to evaluate the composition of the extracts obtained from the roots and leaves ofEutrema japonicumcultivated in Poland. For this purpose, LC-DAD-IT-MS and LC-Q-TOF-MS analyses were used. The results revealed the presence of forty-two constituents comprising glycosinolates, phenylpropanoid glycosides, flavone glycosides, hydroxycinnamic acids, and other compounds. Then, the resultant extracts were subjected to an assessment of the potential cytotoxic effect on human colon adenocarcinoma cells, the effect on the growth of probiotic and intestinal pathogenic strains, as well as their anti-inflammatory activity. It was demonstrated that 60% ethanol extract from the biennial roots (WR2) had the strongest anti-inflammatory, antibacterial, and cytotoxic activities compared to the other samples. Our results suggest that extracts fromE. japonicummay be considered as a promising compound for the production of health-promoting supplements.