Fungal-bacterial biofilms dominate microbial life in streams and small rivers and constitute an important component for the global biogeochemical cycling, including pesticides, and an important source of food for many consumers, yet they remain little explored. Experimental validation of real environmental pollution scenarios is urgently needed to build an accurate prediction and assessment of the responses of fluvial biofilms under pesticide pollution. Tebuconazole (TBZ) pollution in agricultural rivers is a threat for freshwater ecosystems since this triazole fungicide inhibits fungal cell growth by blocking the ergosterol biosynthesis pathway. Our study aims to assess the impact of TBZ on fungi-bacteria-algae biomass, diversity, and interactions in stream biofilms, as well as the role of contaminant pre-exposure history on microbial dissipation of TBZ. Natural biofilms communities were harvested from a Swedish stream and were exposed for 24 days to environmental concentrations of tebuconazole (10 and 100 µg/L) under laboratory conditions. Diversity and community structure of fungi, bacteria and algae were assessed using ITS and 16S amplicon sequencing, pigment and ergosterol HPLC/LC-MS analysis and fatty acids GC-MS analysis.The 16S rRNA gene sequencing will reveal changes in the relative abundance of the main prokaryotic phyla due to TBZ exposure. Results from ITS2 amplicon sequencing will reveal the impact of TBZ in fungal diversity, identify tolerant taxa to the pesticide as well as to infer changes in fungi-bacteria interactions via network analysis. Overall, this study will demonstrate that tebuconazole poses risk for freshwater systems due to its persistence, limited microbial biodegradation and high toxicity to fungi.