Recent evidences hilighted the effect of urban effluent on the structure and functions of dowstream periphytic microbial communities associated to increased tolerance to further chemical stress. Nevertheless, the underlying (molecular) mechanisms of these responses remain poorly understood while usual descriptors provide a partial picture of the phenotype of the communities under chemical stress. In this context, the present study aims to gain knowledge about the molecular/biochemical responses of biofilms under urban stress through the implementation of untargeted metabolomics approach. To do so, following exposure in indoor channels connected to a urban effluent, the metabolomic responses of the biofilms were characterized in parallel of structural and functional responses. First, no significant effects were noted on the photosynthesis, respiration, primary/secondary production, as well as on biomass whereas exposure to the raw effluent led to tolerance acquisition to further chemical stress likely associated to significant shift in the microbial diversity. Then, the metabolomic profiles showed discrepancies between all the tested conditions demonstrating a clear effect of the effluent on the molecular phenotype of the biofilms. Moreover, the clear separation between raw and ultrafiltrated conditions highlighted the potential contribution of the microbes from the effluent in the response. Such pattern paralleled with microbial diversity one. The strong influence of the microbes was further confirmed through HCA showing that control and ultrafiltrated conditions were clustered together.Further trends analysis combined with pathways analysis revealed potential impact on porphyrin and chlorophyll metabolism, among others. Finally, statistical comparison between the metabolomic profiles of raw vs ultrafiltrated conditions highlighted that only 11% down- and 18% up-regulated signals contributed to the tolerance of the biofilm. Overall, this study demonstrates that the metabolomic response is more sensitive than usual descriptors through its ability to discriminate all the experimental conditions. Moreover, our results showed that microbes strongly contribute to these various molecular/biochemical responses, as well as to tolerance acquisition. Further investigations are ongoing to confirm the identity of the candidate metabolites and pathways and discriminate their link to urban microbes vs chemicals associated to the tolerance acquisition to chemical stress.