Facing global change, periphyton is a relevant model to evaluate the impairment of ecosystem functions in aquatic systems. However, there is still paucity knowledge about the differences in species sensitivity to chemical stress in these communities, which is critical to their structure and functioning. In this context, this study aims to fill this gap at the biochemical level through the comparison of the molecular phenotype of cyanobacteria, a green alga, a diatom and their co-culture facing a chemical stress. To this end, prior and following a seven days exposure to atrazine, S-metolachlor and their mixture at three concentrations (10, 100 and 1000 µg/L), the metabolome of the three species and their co-culture were characterized using UPLC-HRMS-based untargeted metabolomics. The comparison of the metabolic fingerprints highlighted that prior to exposure, the metabolism of the diatom and green algae were more similar than that of the cyanobacteria, while the co-culture was closer to the diatom metabolism. Both herbicides caused a shift in the metabolic fingerprint of the three species and the consortia, but the modulated metabolic features and/or the intensity of the modulations differed between the species. Moreover, atrazine and S-metolachlor as well as their mixture modulated the metabolome differently for each species, suggesting a potential discrepancy in the toxicity pathways. Further investigations are ongoing to annotate the metabolites and pathways involved in the discrepancies of the molecular phenotypes before and after the exposure. This study will provide new knowledge to better understand species sensitivity differences to chemical stress in periphyton.