Fungicides reaching stream ecosystems have different capacities to adsorb to benthic substrata and thus cause varying effects on benthic microbial communities. We evaluated the potential of a triazole fungicide, tebuconazole (TBZ), to adsorb to submerged leaves and sand and assessed TBZ effects on sand- and leaf-associated microbial communities. An indoor stream channel experiment was designed to test TBZ effects slightly above concentrations measured in agricultural streams (10.7 ± 0.3 μg L−1) on the microbial communities associated with either submerged leaves alone, sand alone or a mixed substratum of leaves + sand over 5 weeks. Weekly samples were taken to determine TBZ effects on the biomass and activity (litter breakdown rate, fungal sporulation rate and extracellular enzyme activities) of fungi and bacteria as well as the dissipation of TBZ in stream channels. TBZ significantly reduced fungal biomass (17–20% relative to controls) but increased bacterial biomass (34–50%) on leaves and sand incubated separately. TBZ also significantly inhibited phenol oxidase activity (36%) in sand but not in leaves. Differences in TBZ effects between leaves and sand communities were not explained by differences between substrata in TBZ adsorption but more likely were related to differences in biomass accrual and structure of the microbial communities. Mixing leaves+sand tended to attenuate TBZ effects on microbial communities, which was probably explained by a greater surface area available for TBZ adsorption to the substrata. This hypothesis is supported by the greater TBZ dissipation in stream channels receiving both leaves and sand. However, leaf and sand mixtures not only diluted but also modified TBZ effects on sand communities (14% increase in fungal biomass and 82% increase in the sporulation of aquatic hyphomycetes). This result suggests that sand could serve as a refuge for fungi during TBZ-contamination episodes. Our study points to the importance of substratum heterogeneity for the ecotoxicological effects of TBZ on agricultural streams and highlights the contrasting responses of fungi and bacteria to contamination by a widespread fungicide.