Phytoestrogens, such as daidzein and genistein in plants are suspected as endocrine-disrupting chemicals (EDCs), because their chemical structures are similar to natural or synthetic estrogens, and have estrogenic activity in vitro and in vivo. An EDC study was carried out on the diets of various animals in vivo. However, many of these diets include phytoestrogens and may already possess estrogenic activity. In this study, we evaluated the estrogenic activity of phytoestrogens (such as daidzin, genistin, daidzein, genistein, coumestrol and equol) and the feed diets for experimental animals, such as fish, amphibians and reptiles, towards human estrogen receptors α (hER-α) and β (hER-β), and the genistein and daidzein content in these diets from HPLC analysis. Coumestrol showed the highest estrogenic activity for hER-α and -β in the -S9 test. Equol showed the highest estrogenic activity for hER-α in the +S9 test. The estrogenic activities of coumestrol, equol and genistein were approximately one hundred to two thousand times higher than that of daidzein. Many of these compounds showed higher compatibility with hER-β than with hER-α. A diet for fish from soybean was indicated to contain the highest amounts of genistein and daidzein. Moreover, this fish diet had the highest estrogenic activity for hER-α and -β. The estrogenic activity was found with hydrolysis by β-glucuronidase, showing higher compatibility with hER-β than with hER-α. In addition, correlation between the contents of genistein and estrogenic activities in the diets was found, with the exception of part of the diet. Therefore, this indicates that the genistein content contributes to the estrogenic activity of the diets. These results suggest that in vivo estrogenic activity might be caused by the diet provided to an experimental animal, indicating the necessity for more careful selection of the feeding diet and measurement of estrogenic substances when performing an in vivo screening assay for EDCs.