Temperature-dependent chemical toxicity has become a crucial issue taking into consideration that lakes, especially shallow waterbodies, are impacted by climate change worldwide. In this study, we are looking for an answer to what extent standard ecotoxicity assays being performed under constant and relatively low temperatures are capable of predicting the chemical risk posed by pesticides. Lemna minor test plants were exposed to glyphosate in concentrations in the range of 25, 50, 100, 200, and 400 μg/L at temperatures 10, 15, 20, 25, 30, and 35 °C. Two peaks appeared when growth inhibition was assessed; lower concentrations elucidated higher inhibition, at 20 °C, while higher concentrations were found at a higher temperature of 30 °C. The toxic effect experienced at 20 °C indicates that reported PNEC values cannot be sufficient to protect non-target aquatic species in certain environmental scenarios. In addition to growth inhibition, phytotoxicity was also assessed based on peroxidase (POD) concentrations. In general, POD showed greater sensitivity, already showing a response at the lowest temperature tested, 10 °C. Decreased POD activity was detected in the temperature range of 10–30 °C, most probably indicating damage to cell and plasma membranes.