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Agrochemicals can reach freshwater bodies by drift, leaching, or runoff, where they constitute complex mixtures. Given that glyphosate and cypermethrin are within the most worldwide used pesticides, they are likely to co-occur in freshwater bodies. The aim of this study was to analyze the interaction between glyphosate and cypermethrin formulations on the cladoceran Ceriodaphnia dubia (Richard 1894) through an acute toxicity test and on a zooplankton assemblage through a mesocosm (30 L) experiment. The 24-h LC50 of both isolated pesticides and their equitoxic mixture was obtained for C. dubia. The mesocosm was performed by exposing a zooplankton assemblage to both pesticides isolated and in combination. The acute toxicity of the equitoxic mixture in C. dubia was 3 and 4 times higher than the isolated toxicity of glyphosate and cypermethrin, respectively. The total toxic units of the mixture were 0.53, indicating a synergistic interaction. In the mesocosm experiment, both pesticides also interacted causing a synergistic negative effect in Cladocera and Copepoda abundances. No interactions between pesticides were found for Rotifera; therefore, the mixture effect was considered additive. It is suggested to continue analyzing pesticide mixture effects on the basis of complementary scales of analysis to reach more environmentally relevant information.