L-(+)-ergothioneine has antioxidant and anti-inflammatory properties in vitro and in vivo and has uses as a dietary supplement and as an ingredient in foods, cosmetics, and as a pharmaceutical additive. The clastogenic potential and mutagenic of ergothioneine were assessed in vitro and in vivo. Ergothioneine concentrations up to 5000 μg/mL, with and without metabolic activation, was tested in the chromosome aberration assay with CHL cells and found not to induce structural chromosome aberrations. In the in vivo mammalian erythrocyte micronucleus test, ergothioneine was administered orally to male mice at doses up to 1500 mg/kg for potential genotoxic activity. No increase in the frequency of micronucleated polychromatic erythrocytes was observed. Overall, ergothioneine was not genotoxic in these studies and provides additional experimental evidence supporting the safety of its use as a potential dietary supplement.