Achillea species are widely distributed medicinal plant throughout the world and have been used since ancient time. There are many reports about the traditional use and highly bioactive compounds of these plants. Therefore, it is fundamental to study in detail the chemical composition and biological activities of Achillea species. Thus, the objective of this study was to determine for the first time the effects of methods/solvents on biological activities (antioxidant, antimicrobial, and anti-urease) of Achillea nobilis L.subsp. neilreichii (Kerner) Formanek 15 extracts obtained from different extraction methods/solvents and then to analysis the phenolic contents of the active extracts. The phenolic contents in ethyl acetate and ethanol extracts, which showed strongest phenolic contents and antioxidant activity were analysed separately by high-performance liquid chromatography with diode array detection and were confirmed by liquid chromatography with quadrupole time-of-flight mass spectrometry using targeted mass spectra techniques with accurate mass. The active phenolic compounds were identified as dicaffeoylquinic acid, luteolin-7-O-glucoside, orientin, vitexin, luteolin, quercetin-methyl-ether, isorhamnetin-glucoside in ethyl acetate extract; chlorogenic acid luteolin-7-O-glucoside, quercetin-methyl-ether, luteolin, orientin, vitexin, dicaffeoylquinic acid, apigenin-6,8-di-C-glucoside, luteolin hexoside hexoside in ethanol extract. As a result of this study, dicaffeoylquinic acid, isorhamnetin-glucoside, chlorogenic acid, and apigenin-6,8-di-C-glucoside compounds were analysed for the first time in Achillea nobilis L. subsp. neilreichii (Kerner) Formanek. Therefore, it is thought that ethanol and ethyl acetate extracts can be used as a natural source in the future for food and pharmaceutical industry.