Herbicides may affect fish at different stages of development, such as gamete, embryonic early development, and mature adult stages. Sperm, embryos, and adults of Astyanax altiparanae were used to evaluate the detrimental effects of three herbicide formulations: Roundup Transorb®, Arsenal® NA, and Reglone®. The LC50 values for adults using glyphosate ae and imazapyr ae were 3.14 mg/L and 4.59 mg/L, respectively, while for diquat ai, the LC50 was higher than the greater concentration (28 mg/L) used in the experiment. For the initial stages of embryo development, the LC50 values were 16.515 mg/L glyphosate ae, 9.327 mg/L imazapyr ae, and 1.084 g/L diquat ai. Inhibition of sperm motility was observed at concentrations of 252 mg/L for glyphosate ae, 137 mg/L for imazapyr ae, and 11.3 g/L for diquat ai. Sperm viability measurements were taken at 101 mg/L for glyphosate, 34.6 mg/L for imazapyr ae, and 1.51 g/L for diquat ai, with 63.6%, 65.1%, and 62.9% sperm viability, respectively, in comparison with 87.5% detected in the control without herbicides. Astyanax altiparanae exhibited different sensitivities to the herbicide formulations investigated in different phases of development. Roundup Transorb® is a more toxic herbicide for adult individuals, while Arsenal® NA is the most toxic for early embryonic development and sperm motility. Reglone® showed the lowest toxicity for adults, early embryonic development, and sperm compared to Roundup Transorb® and Arsenal® NA. Astyanax altiparanae may be considered an emerging fish model for toxicological studies for the neotropical region due to its wide distribution and biological characteristics.