Epoxidized fatty acids are generated during food processing and detected in various lipid- and oil-containing foods. Although compounds with an epoxide structure have high reactivity and there is increasing concern about their potential toxic effects such as genotoxicity and cellular damage, information regarding the toxicity of epoxy fatty acids is largely unknown. Therefore, we conducted three in vitro genotoxicity studies (bacterial reverse mutation assay, in vitro micronucleus test, and p53R2-dependent luciferase reporter gene assay) and HepG2 cytotoxicity assays for epoxyoleic acid (EOA) and diepoxylinoleic acid (DELA). The in vitro genotoxicity results of EOA and DELA were uniformly negative. In the cytotoxicity assay, EOA and DELA induced weak cytotoxicity at high concentrations, but the effect was similar to those of oleic and linoleic acids at low concentrations. Considering the existing toxicity information on epoxidized soybean oil, which is a similar compound, there might be little concern concerning the health effects of epoxy fatty acids at low concentrations.