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Background and Aims: Scorzonera ketzkhowelii Sosn. ex Grossh. (Asteraceae) was reported as a new species in the Flora of Turkey in 2010. However, there have been no prior biological or chemical investigations conducted. Methods: This study aims to investigate the antimicrobial activities of the petroleum ether, dichloromethane, ethyl acetate, and n-butanol fractions derived from S. ketzkhowelii, and determination of secondary metabolites by Liquid Chromatography with High Resolution Mass Spectrometry (LC-HRMS) analysis on the most active antimicrobial fractions. Results: Notably, all of the fractions demonstrated antimicrobial activity against selected microorganisms, with the dichloromethane fraction of the aerial part exhibiting a higher inhibition of microbial growth compared to the other extracts. The findings of our study revealed the presence of several phenolic compounds in the dichloromethane fractions from both the aerial and subaerial parts, including 3,4-dihydroxybenzaldehyde, salicylic acid, dihydrocaffeic acid, caffeic acid, caffeic acid phenethyl ester, chlorogenic acid, quercetin, hyperoside, naringenin, apigenin, hispidulin, hispidulin 7-glucoside, chrycin, emodin, and carnosic acid. Furthermore, dichloromethane fraction of the aerial parts contained additional phenolic compounds such as homogentisic acid, verbascoside, (+)-trans taxifolin, apigenin 7-glucoside, luteolin, orientin, and chrysoeriol. Conclusion: The plant's demonstrated antimicrobial attributes and the diverse range of phenolic compounds it contains present it as a promising subject for continued research and potential applications within the realms of medicine and pharmaceuticals. Further exploration of its bioactivity and potential health benefits may reveal novel avenues for its practical utilization. [ABSTRACT FROM AUTHOR]