Fatty acids are essential elements for the structure of biological membranes and for the storage of metabolic energy. They are used as a source of energy by metabolism at each trophic level, making fatty acids biochemically and physiologically important compounds (Neves et al. 2015). In the trophic chain, many fatty acids are only synthesized by microalgae and bacteria before being transferred via herbivorous invertebrates to fish and ultimately to humans (Arts et al. 2001). For example, highly unsaturated fatty acids (HUFA) such as eicosapentaenoic acid (EPA; C20:5n3), can not be synthesized de novo or in insufficient proportions by animals (Saito and Aono 2014). That is why fatty acid analysis is commonly used to study trophic interactions in food chains. Generally, microalgae with a high proportion of EPA, such as diatoms, are an excellent source of food for animals but the concentrations of these different fatty acids can vary according to the stage of growth of the organism and according to different environmental parameters including pesticide exposure (Brett et al. 2006, Robert et al. 2007, Burns et al. 2011, Filimonova et al. 2016). Moreover, for several years, the intensive use of pesticides caused many problems to the environment, making pesticides major pollutants of aquatic ecosystems (Aydinalp and Porca 2004). The aim of this study is to investigate the impact of 3 pesticides on diatom's fatty acids. To address this issue, a model freshwater diatom (Gomphonema gracile) was exposed to three herbicides, with three different cellular targets, at environmentally relevant and higher concentrations (diuron and S-metolachlor, C1= 1 µg/L and C2= 10 µg/L; glyphosate, C1= 5 µg/L and C2= 50 µg/L). After a 1-week exposure, fatty acid compositions of diatoms were determined by gas chromatography. In comparison with control samples the percentage of 1) polyunsaturated fatty acids (PUFA) decreased with S-metolachlor contamination (C2); 2) saturated fatty acid (SFA) and monounsaturated (MUFA) decreased with diuron and glyphosate exposure (C2). The decrease of PUFA is a direct impact and can be explained by the mode of action of S-metolachlor which inhibits elongases. Concerning diuron and glyphosate, the decrease of SFA and MUFA can reflect an indirect effect, which can be explained by the mode of action of these two pesticides which respectively blocks electron transfer in photosynthesis, and inhibits the synthesis of aromatic aminoacids.