Fatty acids (FA), which are the main components of storage and structural lipids, are crucial for the maintenance of membrane fluidity and play a central role in metabolic energy storage. Pesticide impacts on FA profiles have been documented in marine microalgae, but remain understudied in freshwater diatoms. In the same way, no study has focused on the impact of herbicides on their lipid content. The main aims of this study were to: 1) compare the responses of FA profiles and “classical descriptors” in order to evaluate their complementarity, 2) estimate the effect of herbicides on lipid content and composition of one freshwater diatom and finally, 3) examine the potential of 20:5n3 as a biomarker of herbicide pollution. To address these issues, Gomphonema gracile and Nitzschia palea were exposed during seven days to diuron and S-metolachlor at 10 μg.L-1. The results demonstrated higher toxicity of diuron compared to S-metolachlor at 10 μg.L-1 on all descriptors studied. FA responded to diuron contamination conversely to pigment content and permitted to document energy content variations in diatoms subjected to herbicide stress conversely to “classical descriptors”. Total lipid composition was impacted by diuron with a notable decrease of storage lipids. Diuron affected the content of 20:5n3 in lipids involved in photosynthesis. Based on previous studies and our current findings, it is suggested that 20:5n3 content in diatoms could be a good biomarker of diuron pollution in freshwaters.