Diatoms are commonly used as bioindicators of trophic and saprobic pollution in rivers. However, more knowledge is needed concerning their sensitivity to toxicants such as agricultural herbicides. In this study, seven species of periphytic diatoms were isolated from the Morcille River (Beaujolais area, France) which presents a streamward contamination gradient by pesticides and particularly diuron. The sensitivity of these species to diuron was assessed through ecotoxicological tests based on short-term growth inhibition of monospecific cultures. After application of an appropriate toxicological model, EC50 were determined and the species were ranked according to their tolerance. EC50 values ranged from 4.5 to 19 μg L−1. Finally, the results were related to field periphyton samples from an upstream and a downstream site in order to check if variations in specific relative abundance between sites are consistent with differences in tolerance to diuron. Species distribution between sites was only partially in accordance with toxicological results suggesting that other factors (toxic or trophic) have an important influence on diatom communities in the river. Nevertheless, diatoms showed their potential to indicate water contamination by pesticides and toxic indices could be developed in complement to existing trophic indices