One of the major threats to freshwater biodiversity is water pollution including excessive loads of nutrients, pesticides, industrial chemicals, and/or emerging contaminants. The widespread use of organic pesticides for agricultural and nonagricultural (industry, gardening, etc.) purposes has resulted in the presence of their residues in various environments, including surface waters. However, the contribution of pesticides to the deterioration of freshwater ecosystems (i.e., biodiversity decline and ecosystem functions impairment) remains uncertain. Once in the aquatic environment, pesticides and their metabolites can interact with microbial communities, causing undesirable effects. The existing legislation on ecological quality assessment of water bodies in Europe is based on water chemical quality and biological indicator species (Water Framework Directive, Pesticides Directive), while biological functions are not yet included in monitoring programs. In the present literature review, we analyze 20 years (2000–2020) of research on ecological functions provided by microorganisms in aquatic ecosystems. We describe the set of ecosystem functions investigated in these studies and the range of endpoints used to establish causal relationships between pesticide exposure and microbial responses. We focus on studies addressing the effects of pesticides at environmentally realistic concentrations and at the microbial community level to inform the ecological relevance of the ecotoxicological assessment. Our literature review highlights that most studies were performed using benthic freshwater organisms and that autotrophic and heterotrophic communities are most often studied separately, usually testing the pesticides that target the main microbial component (i.e., herbicides for autotrophs and fungicides for heterotrophs). Overall, most studies demonstrate deleterious impacts on the functions studied, but our review points to the following shortcomings: (1) the nonsystematic analysis of microbial functions supporting aquatic ecosystems functioning, (2) the study of ecosystem functions (i.e., nutrient cycling) via proxies (i.e., potential extracellular enzymatic activity measurements) which are sometimes disconnected from the current ecosystem functions, and (3) the lack of consideration of chronic exposures to assess the impact of, adaptations to, or recovery of aquatic microbial communities from pesticides. Environ Toxicol Chem 2023;00:1–22. © 2023 SETAC