Schizonepeta annua (Pall.) Schischk. is an endemic annual plant from the Lamiaceae family and it has been employed to cure tracheitis in traditional herbal medicine. Its essential oil exhibited a strong antimicrobial and antioxidative effect. Next, high-performance thin-layer chromatography-bioautography was applied for investigation of the bioactive compounds of S. annua, and gas chromatography-quadrupole time-of-flight mass spectrometry was used to perform subsequent targeted identification of compounds. Three active components were characterized, and two of them were tentatively identified as thymol and carvacrol. S. annua has the potential to be a good alternative for synthetic disinfectants and antioxidants.