Studies of the South Saskatchewan River confirmed that N,N‐diethyl‐m‐toluamide (DEET) is ubiquitous at 10 to 20 ng/L, whereas in effluent‐dominated Wascana Creek, levels of 100 to 450 ng/L were observed. Effects of DEET exposure were assessed in microbial communities using a wide variety of measures. Communities developed in rotating annular reactors with either 100 or 500 ng/L DEET, verified using gas chromatography‐mass spectrometry analyses. Microscale analyses indicated that both DEET concentrations resulted in significant (p < 0.05) declines in photosynthetic biomass, whereas bacterial biomass was unaffected. There was no detectable effect of DEET on the levels of chlorophyll a. However, pigment analyses indicated substantial shifts in algal–cyanobacterial community structure, with reductions of green algae and some cyanobacterial groups at 500 ng/L DEET. Protozoan/micrometazoan grazers increased in communities exposed to 500 ng/L, but not 100 ng/L, DEET. Based on thymidine incorporation or utilization of carbon sources, DEET had no significant effects on metabolic activities. Fluorescent lectin‐binding analyses showed significant (p < 0.05) changes in glycoconjugate composition at both DEET concentrations, consistent with altered community structure. Principal component cluster analyses of denaturing gradient gel electrophoresis indicated that DEET exposure at either concentration significantly changed the bacterial community (p < 0.05). Analyses based on 16S ribosomal RNA of community composition confirmed changes with DEET exposure, increasing detectable beta‐proteobacteria, whereas actinobacteria and acidimicrobia became undetectable. Further, cyanobacteria in the subclass Oscillatoriophycideae were similarly not detected. Thus, DEET can alter microbial community structure and function, supporting the need for further evaluation of its effects in aquatic habitats. Environ Toxicol Chem 2019;38:2414–2425. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC