5-Phenylethenylbenzofuroxans have displayed in vitro and in vivo activity against Trypanosoma cruzi, the etiologic agent of American Trypanosomiasis. On the basis of benzofuroxans pre-clinical studies we evaluated the potential of six 5-phenylethenyl derivatives to induce cytotoxicity, mutagenicity and genotoxicity using different in vitro models. Cytotoxic effects were evaluated using a set of cells, mammal pre-monocytic macrophages, V-79 lung fibroblast from Chinese hamster, and colorectal adenocarcinoma Caco-2 cells, in the MTT viability assay. Mutagenicity was tested in the Ames assay using Salmonella typhimurium TA98 strain with and without metabolic activation by S9-rat liver homogenate. The genotoxic potentials were evaluated with the alkaline single cell gel electrophoresis (comet assay) in V-79 cells. In view of the Ames test results we study whether the main mammals' phase I metabolites, the corresponding o-nitroanilines, are involved in the mechanism of mutagenicity. These metabolites are produced by NADPH-dependent enzymes in cytosol and by xanthine oxidase and cytochrome P450 in microsomes from rat liver. Among them, the electronic property of phenyl substituent seems to be responsible for this effect. It could be pointed out that the equimolecular mixture of compounds 1 and 2 (5E- and 5Z-(2-phenylethenyl)benzofuroxan, respectively) could be used in further clinical studies as anti-T. cruzi drug.