Freshwater ecosystems are presently under a range of increasing pressures, some of which being directly due to anthropogenic activities such as agricultural practices or urban activities. Biofilms are communities of microorganisms (bacteria, algae, protozoa, fungi) and meiofaune integrated in an extracellular polysaccharide matrix fixed on immersed substrates. At the base of the trophic chain, biofilms play a key role in aquatic ecosystems. Due to their capacity to rapidly react to contamination, they are seen as efficient bioindicators and are increasingly used for water quality evaluation. Diatoms in particular dominate the algal biomass of biofilms and are routinely used in ecotoxicology to assess the impact of contaminations on freshwater ecosystems. Traditional ecotoxicology approaches rely on quantitative concentration response relationships highlighted on a batch of biological endpoints such as mortality, reproduction, growth and development, but all those approaches are materially and time expensive. We believe that metabolomics can enhance our knowledge of contaminant mode of actions, resulting in the identification of stress induced biomarkers. The present study aims to highlight metabolome changes in the freshwater diatom Nitzchia palea exposed to an herbicide, the diuron (0.3 µg/L and 3 µg/L), during 4 hours. An extraction protocol as well as a non-targeted metabolomic approach using high resolution mass spectrometry (LC-TOF-MS) was developed in order to decipher the impact of contamination on a metabolic level. Ongoing works will be dedicated to optimize both extraction and analysis methods to determine relevant biomarkers for risk assessment.