This study aimed to examine the antibacterial, antioxidant, and enzyme activity of Centaurea hyalolepis, as well as the total phenolic and carotenoid contents, and volatile and phenolic components. Gas chromatography-mass spectrometry was used for the volatile component analysis of C. hyalolepis, while liquid chromatography-mass spectrometry was used for the phenolic component analysis. Furthermore, the antimicrobial activity was evaluated using disc diffusion and microdilution techniques. The antioxidant capacity was investigated in vitro using four distinct methods (2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, cupric ion-reducing antioxidant capacity, and N,N-dimethyl-p-phenylene diamine). Additionally, both in vitro and in silico studies were carried out to determine the inhibitory effect on xanthine oxidase and elastase. Consequently, it was determined that C. hyalolepis has antioxidant properties and is a highly efficient antibacterial agent against Bacillus megaterium, Pseudomonas aeruginosa, Bacillus subtilis subsp. spizizenii, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Klebsiella aerogenes, and Candida albicans. Additionally, C. hyalolepis contains many volatile compounds, such as 2-methyl propanoic acid, 3,5-octadien-2-one, 2,6-dimethyl cyclohexanol, hexanal, pentanal, anethole, and beta-cyclocitral. It also has a large number of phenolic components, including chlorogenic acid, apigenin 7-glucuronide, quinic acid, and 4-hydroxybenzoic acid. Moreover, C. hyalolepis was shown to inhibit xanthine oxidase and elastase.