Mixture risk assessment predictions have rarely been confronted with biological changes observed in the environment. In this study, long-term monitoring of a European great lake, Lake Geneva, provides the opportunity to assess to what extent the predicted toxicity of herbicide mixtures explains the changes in the composition of the phytoplankton community next to other classical limnology parameters such as nutrients. To reach this goal, the gradient of the mixture toxicity of 14 herbicides regularly detected in the lake was calculated using concentration addition and response addition models. A temporal gradient of toxicity was observed which decreased from 2004 to 2009. Redundancy analysis and partial redundancy analysis showed that this gradient explains a significant portion of the variation in phytoplankton community composition with and without having removed the effect of all other co-variables. Moreover, species that are significantly influenced, positively or negatively, by the decrease of toxicity in the lake over time are highlighted. It can be concluded that the herbicide mixture toxicity is one of the key parameters to explain phytoplankton changes in Lake Geneva.