The contamination of aquatic systems by pesticides in agricultural watersheds can directly or indirectly impact the structure and functioning of algal and microbial communities. In small rivers, these microorganisms are organised in biofilm (or periphyton) that can be used as an indicator of ecosystem impairments. The quantification of pesticide concentration in biofilm may be a better assessment of the real exposure of periphytic microorganisms to pesticides. We present here the results of (i) the development and validation of an analytical method for the measurement of 15 pesticides in biofilm and (ii) a laboratory experiment aimed at evaluating sorption kinetics of pesticides in biofilm, and measuring associated effects. The analytical development was performed by means of experimental microcosms design. This methodology allowed to identify main factors affecting the performance of the method, then to efficiently optimize analytical recoveries. Pesticides were extracted from dried biofilm with acetone under pressurized liquid extraction. The organic extract was then analysed by ESI-LC-MS-MS. Recoveries ranged from 52 to 120% (RSD below 15%, n=10) and quantification limits varied between 15 and 350 ng/g dry wet, according to the pesticide. Sorption kinetics in biofilm were evaluated under controlled hydrodynamic conditions. Biofilm was collected after different contact times (0 to 24 hours) with spiked pesticide solutions. Diuron, azoxystrobine and dimetomorph were adsorbed after 15 min, whereas fenitrotion and procymidone were never quantified in biofilms. In parallel, we observed that some herbicides induced an inhibition of the photosynthesis reaction, reflecting therefore the physiological state of these communities, when subjected to a chemical pressure.