Thymoquinone is a popular health-promoting antioxidant supplement, but it may induce toxicity to cells and organs because of its propensity to promote oxidation of biomolecules under some conditions. Furthermore, as hydroquinones have been found to exhibit more potent antioxidant and prooxidant activities than their parent quinones, the reduced metabolite thymohydroquinone may have stronger effects than thymoquinone. In this study, the antioxidant and prooxidant activities of thymoquinone and thymohydroquinone were assessed to determine whether they both act as antioxidants and induce oxidative damage to biomolecules as do other quinones. Using ESR spectroscopy, we demonstrated that thymohydroquinone exhibits more potent antioxidant activity than does thymoquinone. In addition, thymohydroquinone was found to act as a prooxidant to induce oxidative damage of isolated plasmid DNA in the presence of free Cu²⁺ or Fe²⁺–ethylenediaminetetraacetic acid (EDTA). Interestingly, the prooxidant effect of thymohydroquinone in the presence of Fe²⁺ was not observed in the absence of EDTA. Thymohydroquinone thus was demonstrated to have two biologically relevant activities: as an antioxidant and a prooxidant.