Orthodontic brackets release ions that can be reabsorbed in the oral mucosa, potentially causing complications, including cytotoxic effects and mutagenic alterations. The aim was to evaluate the genotoxicity induced by orthodontic appliance alloys in cultures of human gingival fibroblasts by comet assay. Eluates were obtained from the following brackets alloys: EconoLine (SS: stainless steel), MiniMirage (Ni-Ti: nickel-titanium), Nu-Edge (Co-Cr: cobalt-chromium), In-Vu (PC-polycrystals (PC) aluminum oxide), and Monocrystal IZE (monocrystalline (MC) aluminum oxide). Each bracket was sterilized and exposed to a corrosive process for 35 days. The obtained eluates were tested for genotoxicity of human gingival fibroblasts (HGFA) by the alkaline comet assay. All study groups showed genotoxic effects; there was a significant difference (p<0.0001) among groups. The eluates obtained from Ni-Ti showed a 16-times greater genotoxic effect. There were differences in genotoxicity after comparing the Ni-Ti with SS (p<0.01) and Co-Cr brackets (p<0.001). The ceramic was more genotoxic than metallic brackets (SS and Co-Cr), but less than the Ni-Ti. This in vitro model will be useful for further study of early DNA damage caused by brackets and other biomaterials used in the oral cavity before their introduction into the clinical setting.