Objective The present study aimed to compare the polyphenol content, total phenolic, total flavonoid, antioxidant and antimicrobial activity of the extracts obtained from Centaurea amaena Boiss. & Balansa and Centaurea aksoyi Hamzaoğlu & Budak. Methods Both species were subjected to maceration, Soxhlet and ultrasonication extractions with methanol in order to macerated (ME), Soxhlet (SE) and ultrasonicated (UE) extracts. Their phenolic profiles were qualitatively examined by LC–MS. Their antioxidant activities were determined by phosphomolybdenum, β-carotene bleaching and DPPH assays. Agar diffusion and broth dilution methods were carried out to find the antimicrobial activity of these extracts against fifteen microorganisms. Results Quercetin, quercetin-3-β-D-glucoside and protocatechuic acid were the main components of the both extracts obtained by Soxhlet extraction. The highest phenolic and flavonoid contents were found in the UE for both species. All the extracts exhibited good total antioxidant and DPPH radical scavenging activity. UE obtained from C. amaena showed the highest antioxidant activity with the highest phenolic and flavonoid contents. The antibacterial activity of UE obtained from C. amaena was better than other extracts and antibacterial activity of C. amaena was also better than C. aksoyi. Discussion This study confirms that ultrasonic extraction may be an ideal, simple and rapid method to obtain polyphenol-rich extracts have antioxidant as well as antibacterial activity from both Centaurea species especially from C. amaena.