Carvacrol and thymol are phenolic compounds with similar structures isolated from many aromatic plants, and have been demonstrated to exert multiple pharmacological effects. The metabolic and pharmacokinetic behaviour of thymol and carvacrol has received much attention. Carvacrol and thymol have been demonstrated to undergo phase I metabolism such as hydroxylation reaction. However, drug-metabolizing enzymes involved in this process remain unclear. Given that cytochrome P450s (CYPs) are involved in most phase I metabolism, the aim of the present study was to investigate the role of CYPs in the metabolism of thymol and carvacrol. After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol. All these results remind the researchers that special attention should be paid on pharmacokinetic and clinical outcomes when thymol or carvacrol was co-administrated with other compounds mainly undergoing CYP2A6-mediated metabolism.