The environmental impact of diffuse pesticide pollution was investigated during two seasons (end of spring and winter) on natural river biofilms, both in a pristine and in two contaminated stations in the Morcille river. Prokaryotic and eukaryotic community composition was assessed by Denaturing Gradient Gel Electrophoresis DGGE, whereas microalgal community composition was described by High Pressure Liquid Chromatography (HPLC) pigment analysis. The sensitivity of microphytobenthos to Diuron was investigated at the laboratory by photosynthesis short-term inhibition assays. Based on DGGE and on HPLC analyses, spatial changes in prokaryotic and eukaryotic species composition were found both in spring and in winter. The structure of the communities was significantly different in samples from contaminated areas, compared to less or non-contaminated ones, without leading to a decrease in biodiversity. Community tolerance towards Diuron, based on EC50 values for photosynthesis, were significantly lower for upstream than for downstream photoautotrophic organisms, whereas pesticide concentration increased from up- to downstream. These results strongly suggest contamination-driven changes in biofilm community structure and in the tolerance of the photoautotrophic community, confirming the Pollution-Induced Community-Tolerance (PICT) hypothesis.