In order to assess the real exposure of biofilm (or periphyton) to pesticides in contaminated aquatic systems, analytical methods were developed to measure pesticide concentrations in surface water and freshwater biofilm. After extraction of (i) biofilm in ultrasonic bath with organic solvents or (ii) water with solid phase extraction technique, the pesticides were quantified by LC-ESI-MSMS. The methods were applied in the field in order to study the spatial and temporal variation of periphyton contamination. Samples of biofilm and water were collected during the spring and summer 2006 in a small stream of the Beaujolais area (France) submitted to a gradient of contamination due to pesticide treatment on vineyards. All the seven studied pesticides were detected and quantified in water but only two were detected in biofilms. Concentrations of pesticides in biofilm generally followed that of the dissolved contaminants in surface water, increasing downstream for both streams and were correlated to treatments. The evolution of pesticide concentrations in water was also evaluated during storm events in order to have a better evaluation of the real exposure of organisms to organic contaminants not only during base flow.