Extensive pesticide use for agriculture can diffusely pollute aquatic ecosystems through leaching and runoff events and has the potential to negatively affect non-target organisms. Atrazine and S-metolachlor are two widely used herbicides often detected in high concentrations in rivers that drain nearby agricultural lands. Previous studies focused on concentration-response exposure of algal monospecific cultures, over a short exposure period, with classical descriptors such as cell density, mortality or photosynthetic efficiency as response variables. In this study, we exposed algal biofilms (periphyton) to a concentration gradient of atrazine and S-metolachlor for 14 days. We focused on fatty acid composition as the main concentration-response descriptor, and we also measured chlorophyll a fluorescence. Results showed that atrazine increased cyanobacteria and diatoms chlorophyll a fluorescence. Both herbicides caused dissimilarities in fatty acid profiles between control and high exposure concentrations, but S-metolachlor had a stronger effect than atrazine on the observed increase or reduction in saturated fatty acids (SFAs) and very long chain fatty acids (VLCFAs), respectively. Our study demonstrates that two commonly used herbicides, atrazine and S-metolachlor, can negatively affect the taxonomic composition and fatty acid profiles of stream periphyton, thereby altering the nutritional quality of this resource for primary consumers.