An HPLC quadrupole time-of-flight (Q-TOF) MS method was developed for the identification of secondary metabolites in Leonurus turkestanicus V.I. Krecz. et Kuprian. Ethanolic and chloroform extracts from the plant's aerial parts were tested. A total of 16 compounds (iridoid glycosides, phenylpropanoids, flavonoids, and nitrogen-containing compounds, as well as diterpene acetate derivatives) were identified and tentatively characterized based on (or using) their retention times and UV and Q-TOF-MS data. Previously reported aucubin (1), 6-deoxy-8-acetylharpagid (2), and stachydrine (13, 15) and homostachydrine isomers (14, 16) were identified, along with a lavandulifolioside isomer (3), verbascoside (4), rutin (5), 3-O-kaempferol rutinoside (6), and an unknown diterpene acetate (8). Compounds 3-6 were detected for the first time in this plant. Additionally. antimicrobial activity was evaluated. No significant differences were found between ethanolic and chloroform extracts of L. turkestanicus; however, the alcoholic extract showed stronger antifungal activity [minimal inhibitory concentration (MIC) of 2.5-5 mg/mL], whereas the chloroform extract showed stronger activity against the tested spore-forming Bacillus species (MIC 1.25-2.5 mg/mL).