Neonicotinoids, a class of neuroactive insecticides chemically similar to nicotine, are now the most widely used pesticides worldwide. Highly water-soluble, approximately 95% of their application disperses into the environment rather than being absorbed by the target crop. As a result, neonicotinoids can easily be transported into streams, rivers, and lakes, posing a significant threat to the health and quality of these ecosystems. Stream biofilms play a crucial role in maintaining aquatic ecosystem function, yet they may be negatively impacted by neonicotinoids. However, the specific effects of these pesticides on biofilms remain largely unknown. We conducted a field experiment to examine the effects of the neonicotinoid insecticide Imidacloprid on stream biofilms. To do this, we deployed contaminant exposure substrata (CES) with three Imidacloprid concentrations—control, 0.01 M, and 0.1 M—in White Clay Creek, an experimental stream in southeastern Pennsylvania. We tested three exposure durations: 5 days, 10 days, and 20 days. After the exposure period, biofilms grown on the substrata were analyzed for biomass, respiration, primary production, and community composition (including both prokaryotes and eukaryotes) using amplicon high-throughput sequencing. The results demonstrated that Imidacloprid inhibits microbial growth and function by (a) suppressing biodiversity, (b) altering community composition, and (c) increasing respiration and photosynthesis. These findings suggest that Cyanobacteria and algae exhibit greater tolerance to pesticide exposure. This study provides critical insights into the mechanisms by which neonicotinoid pesticides negatively impact the environment, particularly their effects on stream biofilms and, consequently, ecosystem processes and function.