In 2000, the EU Water Framework Directive (directive 2000/06/EC) was implemented with the objective of reaching the good ecological status of rivers. 45 chemicals were indexed as priority including 19 pesticides. The biofilm is at the basis of the trophic chain in aquatic environments and considered as an excellent bioindicator for water quality assessment (Edwards and Kjellerup 2013) because of its ability to integrate contamination (Vercraene-Eairmal et al. 2010). In this study, we used a photosynthesis inhibitor herbicide: diuron, one of the priority substances to the EU Water Framework Directive. Previous experiments carried out in order to characterize diuron bioaccumulation in biofilms, with two different exposure concentrations, suggest that pesticide uptake by microorganisms was not proportional to contaminant concentration in the water. For this experiment, we supposed that diuron absorption isotherms are not linear, and in order to confirm this hypothesis, bioaccumulation and toxic impact were simultaneously assessed at the equilibrium. To that aim, mature biofilm previously grown on glass slides during one month was exposed in channels at 8 increasing concentrations of diuron: 0, 1, 5, 10, 25, 50, 75 and 500 µg.L-1 for two hours, with a flow velocity of 2 cm.s-1. Then, Langmuir isotherm equation (Praus et al. 2007) was fitted to the bioaccumulation data. Diuron bioaccumulation in biofilm at equilibrium was not linear. A plateau was reached over 5 µg.L-1 of diuron in the water. This suggested that all absorption sites were saturated, and then diuron concentration in the biofilm became independent of diuron concentration in the water. The fitting of a Langmuir isotherm allowed to estimate a maximal diuron concentration in biofilm at 2073 µg.g-1, and the equilibrium constant at 0.378. Photosynthesis inhibition was correlated (R²=0.75) to diuron concentration in the water. The data didn't clearly highlighted a relationship between bioaccumulation and photosynthesis inhibition. This study establishes that diuron bioaccumulation in biofilm is nonlinear, and allows to calculate the equilibrium constant and maximal amount of diuron potentially accumulated in biofilm. These two constants can be used to further predict diuron concentrations in biofilm from diuron concentration in the water. The coupling of toxicokinetic and toxicodynamic approach provides original information about pesticide behaviour and impact in periphytic microorganisms.