<p>Streams in agricultural areas are susceptible to contamination by various compounds, including pesticides. Although spot water sampling is traditionally used for monitoring pesticide contamination of surface waters, this method may underestimate the presence and bioavailability of certain compounds. To better assess environmental exposure, this study investigates the potential of epilithic biofilm (periphyton) as a biomonitor of pesticide contamination. Biofilms were sampled from several streams in the province of Quebec (East Canada) subjected to “moderate” and “intense” agricultural pressure to determine pesticide accumulation of selected herbicides (atrazine, S-metolachlor, imazethapyr) and insecticides (chlorantraniliprole, clothianidin, thiamethoxam, chlorpyrifos) using an optimized QuEChERS extraction method. Benthic macroinvertebrates were also sampled at certain sites to assess their potential as indicators of pesticide exposure using the same approach as biofilms. High detection frequencies and elevated concentrations of atrazine, S-metolachlor, chlorantraniliprole and chlorpyrifos were observed in periphyton from areas under intense agricultural pressure. Pesticides were more frequently detected in water than in periphyton, except for chlorpyrifos. Relationships between concentrations in water and in periphyton followed a linear model for atrazine, S-metolachlor and chlorantraniliprole. This study highlights the potential of periphytic biofilms as complementary tools for pesticide monitoring, although further laboratory and in situ validation is needed. Macroinvertebrates also accumulated pesticides under intense agricultural pressure, particularly the insecticide chlorantraniliprole, raising concerns about potential risks to invertebrate communities. These findings illustrate the affinity of pesticides for different matrices (water versus periphyton versus invertebrates) and the need for monitoring programs not to rely solely on spot water measurements.</p>