YY technology has been developed in response to the demand of environment-friendly techniques to produce all-male populations in Nile tilapia culture. However, the first part of this technique still requires feminization of XY fry. Use of estrogen mixtures to achieve feminization has never been tested in Nile tilapia, so the aim of this study was to evaluate the effect of different combinations of the three most important estrogens (17a-estradiol E2, diethylstilbestrol DES, and 17a-ethinylestradiol EE2) on sex proportion, growth, and gonadal development. Mixtures evaluated were E2-DES, E2-EE2, DES-EE2, and E2-DES-EE2. No significant differences in growth were observed at the end of the experiment between control fish and fish fed estrogen mixtures. However, final survival was significantly lower (P < 0.05) in fish fed E2-DES and E2-DES-EE2. All estrogen mixture-treated groups produced progenies with a significantly higher (P < 0.001) proportion of males than the predicted 1:1 sex ratio. No significant differences were observed in gonadosomatic index between fish of the control group and fish fed estrogen mixtures. The proportion of deformed gonads was higher in groups fed E2-DES and E2-DES-EE2. The paradoxical masculinization observed in all estrogen mixtures was probably provoked by the interaction of estrogens with androgen receptors or the inhibition of aromatase expression, which resulted in testosterone accumulation and testis development. These results address the need for further research towards understanding the role of steroids in sexual development since these findings add to a short list of studies that report paradoxical effects after using steroids.