In order to characterize the chemical components in three thyme plants from thyme genus and evaluate their bacteriostatic activity and cytotoxicity on human cancer cells, the volatile oil from T. citriodorus, T. mongolicus, and T. vulgaris was extracted by steam distillation and their chemical components were analyzed.A total of 50 compounds were detected, of which the highest thymol content was 37.61%, citral was 11.44%, cis-geraniol was 10.25%, caryophyllene oxide was 5.74%, and thymol methyl ether was 12.45%.The antibacterial activity from these three types volatile oils was evaluted by filter paper method and plate coating method and volatile oil of T. citriodorus was determined as the best for its antibacterial effect compared with others.The minimum bacteriostatic concentration against three strains of bacteria was in the order of Escherichia coli> Fusarium avenaceum > Staphylococcus aureus.The CCK-8 method was used to determine the cytotoxicity of three thyme volatile oils on HepG2 liver cancer cells.The results showed that volatile oil from T. citriodorus had the strongest inhibitory effect on liver cancer cells with an IC₅₀ value of 18.32 μg/mL.The results from this research can provide a theoretical basis for the development and utilization of thyme volatile oil.