Urban wastewater treatment plants (WWTPs) release large amounts of pollutants (e.g. chemicals, microbes) into aquatic environments that may impact exposed organisms. Among these organisms, aquatic biofilms as a complex assemblage of microorganisms with a short life cycle (e.g. microalgae, bacteria, etc.) and by their key role in aquatic ecosystems (e.g. primary production) are relevant and increasingly used to investigate the impact of chemical contamination at the community level. Despite increasing knowledge on the impact of chemical stress on these communities, the underlying (molecular/biochemical) mechanisms remain poorly described while usual descriptors provide a partial picture of phenotype. To tackle this issue, untargeted metabolomics approach is relevant through the simultaneous characterization of chemical exposure and the global response of the whole biofilm. In this context, this study focuses on the characterization of the potential impact of urban (WWTPs) on aquatic periphytic microbial communities by implementing an untargeted metabolomic approach. To do so, colonization/exposure was carried out during 4 weeks at upstream and downstream sites from three WWTPs along a tributary of the Arcachon Basin (Belin Beliet: upstream, Salles: intermediate; Mios: downstream). First, multivariate analyses (PCA and HCA) showed discrepancies in the metabolomics profile between the three WWTPs and between upstream and downtream site of each WWTP. This difference is more marked at the downstream site (Mios), likely because of increasing disturbances along the tributary (e.g detection of pesticides only on the downstream site). Moreover, the strong discrimination between upstream and dowstream sites at Mios suggested that this WWTP is a source of pollutants. At this site, further univariate analysis combined to pathways analysis allowed preliminary annotation (i.e. based only on MS1) that highlighted a potential impact on the respiration and photosynthesis pathways. Further investigations are ongoing to improve the annotation through the analysis of MS² spectra and by using an internal database. Overall, this study confirms the relevance of untargeted metabolomics to highlight potential impact of urban discharges on aquatic microbial communities.