The Morcille River located in the Beaujolais vineyard area (Eastern France), is subjected to strong wine-growing pressure leading to the contamination by a range of herbicides and fungicides of the surrounding fresh water environment. The objective of this study was to evaluate the potential use of passive samplers extracts in order to highlight effects of representative pesticide mixtures on natural biofilm communities with diverse exposure history. Chronic and acute impacts of pesticides in mixture were evaluated using POCIS (Polar Organic Chemical Integrative Sampler) extracts on natural biofilm communities originated from a pesticide contaminated site (downstream site) or a reference site (upstream site). Biofilms were exposed in an artificial channel system to POCIS extracts (PE) or placed in clean water during 13 days in order to model different levels of toxic pressure in relation with a realistic pesticide mixture directly isolated from the field. After 13 days of exposure, significant impacts of PE treatment on growth related parameters and on physiological endpoints were observed. A significant decrease of diatoms density was observed on biofilms exposed to PE compared to non exposed biofilms undependably of biofilm origin. Moreover DW and AFDM were lower for downstream biofilms exposed to PE compared to non exposed biofilms. Taxonomic analyses revealed impacts of mixture of pesticides on diatom composition. Upstream communities exposed or not to PE presented different diatom composition with higher proportion of Nitzschia palea and Eolimna minima for contaminated treatment. Downstream communities exposed or not to PE showed very similar composition at the end of the experiment highlighting the importance of immigration processes in the recovery of diatom assemblages (Morin et al., 2012). In order to evaluate initial tolerance of biofilms to pesticide mixture; acute toxicity tests were realized before the beginning of the channel experiment. Acute exposures revealed a significant higher tolerance for biofilm originated from downstream site compared to biofilms originated from upstream site. This difference of tolerance to PE was related to very different taxonomic compositions in regards to biofilm origin; it reflects adaptation of communities by species selection processes and is an illustration of the PICT concept (Blanck et al., 1988).Our results underline impacts of mixture of pesticides on growth (DW, AFDM and diatoms density), diatom composition, and community tolerance of natural biofilms. This study highlights the potential use of passive sampler extracts combined with acute toxicity tests and chronic exposure experiment in order to evaluate effects of more realistic pesticide mixtures on natural biofilms communities, and then the future and likely applications of such approaches for ecological risk assessment.