Herbicide mixture is used as an alternative to obtain different mechanisms of action acting on weeds, resulting in the frequent presence of pesticides in environmental compartments. As they are products used worldwide, this study evaluated effects of environmentally relevant concentrations of the analytical standards and commercial formulations of the herbicides atrazine (2 µg L− 1) and glyphosate (65 µg L− 1), in isolation and also in mixture (2 + 65 µg L− 1) on the microcrustacean Daphnia magna. Through chronic exposure (21 days) of two generations of organisms, effects on survival and reproductive capacity were observed, as well as responses regarding oxidative stress, determined through the analysis of biochemical biomarkers such as catalase and glutathione S-transferase. In the evaluation of the first generation of test organisms, no significant results related to biochemical biomarkers were observed, only effects over sexual maturation of organisms. However, in the second generation of exposed organisms, changes were observed in all parameters evaluated, with the mixture of herbicide active principles being the treatment responsible for more significant responses (p < 0.05). A statistical difference (p < 0.05) was also observed between analytical standards and commercial formulations, indicating that other components present in the formulations can change the toxicity of the products. Given the difficulty of estimating the effects of mixtures and considering that various stressors are found in the environment, our results support the need to carry out studies that address long-term effects and, above all, that verify what the impacts are across generations, so that the toxicity of products is not underestimated.