Water flow velocity disturbances in biofilms are quite well understood, but the consequences for pesticide dissipation have yet to be addressed. We investigated the capacity of biofilms with different histories of hydrological disturbances [frequent flow velocity increase (FI), sporadic flow velocity increase (SI), and a control with constant flow velocity (C)] to dissipate a cocktail composed of herbicide (terbuthylazine), fungicide (tebuconazole), and insecticide (imidacloprid) molecules. Pesticide dissipation and bioaccumulation by biofilms were measured using two different exposure scenarios: i) an open-flow experiment using stream channels and ii) a batch experiment using culture flasks. The effect of flow velocity disturbances on biofilms was characterized by reduced thickness and bacterial density, but higher surface density compared to the constant flow velocity control. The highest dissipation of pesticides was observed in FI biofilms, and the lowest was observed in C biofilms. However, bioaccumulation was significantly higher in SI biofilms regardless of open flow and/or batch experiments. The more hydrophobic molecules (terbuthylazine and tebuconazole) had greater accumulation than imidacloprid in biofilms, and this was irrespective of the hydrological treatment and/or pesticide-exposure approach. Hydrological disturbances enhance pesticide accumulation in biofilms, but further research is needed to understand pesticide remobilization and downstream exposure during these events.