© 2022, Turkiye Klinikleri. All rights reserved.The objective of this study was to evaluate the biological activities and chemical fingerprint profiles of the extracts obtained from twelve Achillea L. species (A. lycaonica, A. biebersteinii (syn: A. arabica), A. kotschyi subsp. kotschyi, A. schischkinii, A. millefolium subsp. millefolium, A. sintenisii, A. setacea, A. teretifolia, A. wilhelmsii subsp. wilhelmsii (syn: A. santolinoides subsp. wilhelmsii), A. nobilis, A. goniocephala, A. spinulifolia). The antioxidant, enzyme inhibitory and cytotoxic effects were evaluated to investigate their bioactivity profiles. Furthermore, the total flavonoid and phenolic contents were determined and LC-MS/MS analysis was performed to reveal the phytochemical profile of the investigated extracts. A. kotschyi and A. nobilis species were detected to have very high antioxidant potential as well as high total phenolic content (260.00 ± 3.38 and 282.97 ± 3.14 μg of PEs mg extract–1, respectively). According to the LC-MS/ MS results, A. kotschyi and A. nobilis species were found to contain very high concentrations of chlorogenic acid (55812.20 and 46407 μg analyte g extract–1). Besides, the bioactivities and phenolic composition of these species were chemometrically analyzed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) techniques. It has also been determined that Achillea species generally exhibit quite high cytotoxic activity against the HeLa cell line. The studied species showed high urease enzyme activities.