The aim of this study was the evaluation of antioxidant, antimicrobial and cytotoxic activity of three Tanacetum species from Romanian flora and the identification of biologically active compounds in the analysed species. T. vulgare and T. corymbosum showed antioxidant activity in correlation with the total phenolic content of the extracts. All three species exhibited antimicrobial effect against the tested Gram-positive bacteria and fungi, and manifested strong cytotoxic activity against both cancer (HeLa) and healthy (Vero) cell lines. The chemical analyses allowed the identification of common phytosterols and many flavonoids, some reported for the first time: casticin in T. vulgare, eupatilin, casticin, acacetin, quercitrin and isoquercitrin in T. macrophyllum, eupatorin, apigenin and quercitrin in T. corymbosum. It is possible that the numerous flavonoids found in these species to contribute to the pharmacological actions of the extracts.