The aim of this investigation was to evaluate the possible protective activity of Agaricus brasiliensis (=A. blazei sensu Murrill) ethanol extract against thymol-induced DNA damage in human lymphocytes. Before we studied the possible interaction of thymol and A. brasiliensis extract, each component was tested in the comet assay. Thymol significantly increased DNA damage in human lymphocytes at higher concentrations (20, 50, 100, 150, and 200 µg/mL), whereas no genotoxic effect of A. brasiliensis ethanol extract was observed. In simultaneous treatment with thymol (200 µg/mL) and A. brasiliensis ethanol extract (50, 100, 150, and 200 µg/mL), the latter failed to reduce a thymol-induced DNA damaging effect regardless of the applied concentrations. To confirm that thymol induces DNA damage via reactive oxygen species, we performed cotreatment with quercetin. Cotreatment with quercetin (100 and 500 µmol/L) significantly reduced DNA damage caused by thymol (200 µg/mL), indicating that thymol exhibits genotoxicity mainly through induction of reactive oxygen species.