The effects of chemical and physical factors on periphyton structure, diversity and functioning were investigated in an outdoor mesocosm experiment. Stream biofilms were subjected to a pesticide mix (diuron and azoxystrobin) under two different hydraulic regimes. The hydraulic regimes differed by spatial variations of flow conditions (turbulent with high variations vs. laminar with low variations). The effects of the hydraulic regime and pesticides were assessed at the level of the periphytic communities. We focused on the change in the biodiversity of these communities under the two hydraulic regimes, and on the role of these biodiversity changes in case of pesticide contamination. Changes in structural (biomass, cell density), diversity (community composition assessed by PCR-DGGE and microscopic analysis) and functional (bacterial and algal production, sensitivity to the herbicide) parameters were monitored throughout a 2-month experiment. The results showed that exposure to pesticides affected the phytobenthic community targeted by the herbicide, impacting on both its growth dynamics and its primary production. Conversely, the impact of the flow regime was greater than that of pesticides on the non-target bacterial community with higher bacterial density and production in laminar mesocosms (uniform regime). An interaction between flow and pollution effects was also observed. Communities that developed in turbulent mesocosms (heterogeneous regime) were more diversified, as a result of increased microhabitat heterogeneity due to high spatial variations. However, this higher biodiversity did not increase the ability of these biofilms to tolerate pesticides, as expected. On the contrary, the sensitivity of these communities to pesticide contamination was, in fact, increased.