Extensive use of herbicides in agriculture is accompanied by the risk of environmental contamination of aquatic ecosystems. The present study shows the effects of the herbicides chlortoluron and mesotrione on three microalgae species: two chlorophyceae (Pediastrum tetras, Ankistrodesmus fusiformis) and one diatom (Amphora coffeaeformis). The authors calculated the IC50 for one chlorophyceae and the diatom. The order of toxicity (median inhibitory concentration [IC50]) for mesotrione was A. coffeaeformis (13.1 mg/L) > A. fusiformis (56.1 mg/L) and A. fusiformis (0.05 mg/L) > A. coffeaeformis (0.08 mg/L) for chlortoluron. The impact of herbicides applied at 0.2 mg/L was then examined in Erlenmeyer flasks by monitoring for growth, pigment content, and metabolic activity. Algal responses varied widely according to species and herbicide. For example, chlortoluron showed a significant inhibitory effect on the growth of A. coffeaeformis, whereas mesotrione induced an increase in cellular density in A. fusiformis. Other cellular parameters, such as pigment content in P. tetras, were stimulated by both herbicides. The results obtained confirmed that microalgae cultures are clearly affected by acute and chronic exposition to herbicides. Further monitoring should be carried out in the field to assess the impact of sublethal levels of toxicity and the growth-enhancing effects of mesotrione and chlortoluron on natural algae communities.