The herbicides atrazine and glyphosate are used worldwide and their excessive usage results in the frequent presence of these pesticides in environmental compartments. We evaluated the effects of environmentally relevant concentrations of analytical standards and commercial formulations of atrazine (2 µg L-1) and glyphosate (65 µg L-1), isolated and in mixture (2 + 65 µg L-1) on the microcrustacean Daphnia magna. Through chronic exposure (21 days) of two generations, we observed effects on survival, reproductive capacity and responses of the antioxidant defense system (catalase) and biotransformation system (glutathione S-transferase). The survival of organisms was affected in the second generation (F1) with a mortality of 17% in the mixture of commercial formulations treatments. In the evaluation of the first generation (F0) we observed only effects on sexual maturation of organisms, while in the F1, changes were observed in all parameters evaluated. A statistical difference (p < 0.05) was also observed between the analytical standards and the commercial formulations for all parameters evaluated, indicating that other components present in the formulations can change the toxicity of products. We suggest that atrazine can modulate toxicity when mixed with glyphosate, as the standard analytical atrazine and mixture of analytical standards results were similar in most parameters. Given the difficulty in estimating effects of mixtures and considering that various stressors are found in the environment, our results support the need to carry out long-term studies and, above all, to verify what are the impacts across generations, so that the toxicity of products is not underestimated. Graphical abstract.