Abstract Extensive pesticide use for agriculture diffusely pollutes 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. To determine the effects of these two herbicides on river ecosystems, we conducted a 14-day laboratory experiment to expose river periphyton to a wide range of atrazine and S-metolachlor concentrations. The response of periphyton was evaluated using ecologically relevant endpoints including chlorophyll a fluorescence and fatty acids composition. Results showed that atrazine disrupted photoautotroph biomass measured by 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 observe increase or reduction in saturated fatty acids (SFAs) and very long chain fatty acids (VLCFAs). Our study demonstrates that two commonly used herbicides, atrazine and S-metolachlor, can negatively affect the composition and fatty acid profiles of stream periphyton, thereby altering the nutritional quality of this resource for primary consumers.