Facing increasing pollution of aquatic ecosystems, one major challenge is to better understand how organisms respond to chemical stress in order to gain knowledge on the resilience of these ecosystems and associated services. To this end, untargeted metabolomics is a method of choice which allows to provide a comprehensive picture of both exposure and effect. Also, its application to aquatic microbial communities, which play a key role in aquatic systems, could improve the ecological relevance of current ecotoxicological approaches through the discovery of ecosystemic biomarkers. Thus, we investigate the responses of aquatic biofilms to chemical stress by using high-resolution mass spectrometry based metabolomics. Our approach consists on the combined measurement of molecular, physiological and structural descriptors following exposure in controlled conditions and/or colonization/exposure on the field. Further chemometric methods help to identify metabolites and associated signalling pathways and link them to structural and functional changes. The specificity of these molecular makers regarding chemical stress and confounding factors can be then investigated through exposure under controlled conditions. Our research finally aims to gain knowledge on the molecular mechanisms involved in the alteration of key ecosystem functions and processes. Since urban activities represent a threat to the environment, we recently investigated the impact of wastewater effluents on microbial communities through colonization/exposure in channels connected to the effluents. Structural (microbial composition), physiological (photosynthesis, respiration) and metabolomic responses have been acquired and are under interpretation. Our approach and the results of this study will be presented.