Abstract Aquatic ecological risk assessment of fungicides in Europe under Regulation 1107/2009/EC does not currently assess risk to non-target bacteria and fungi. Rather, regulatory acceptable concentrations based on ecotoxicological data obtained from studies with fish, invertebrates and primary producers (including algae) are assumed to be protective to all other aquatic organisms. Here we explore the validity of this assumption by investigating the effects of a fungicide (tebuconazole) applied at its “non-microbial” HC5 concentration (the concentration that is hazardous to 5% of the tested taxa) and derived from acute single species toxicity tests on fish, invertebrates and primary producers (including algae) on the community structure and functioning of heterotrophic microbes (bacteria and aquatic fungi) in a semi-field study, using novel molecular techniques. In our study, a treatment-related effect of tebuconazole (238μg/L) on either fungal biomass associated with leaf material or leaf decomposition or the composition of the fungal community associated with sediment could not be demonstrated. Moreover, treatment-related effects on bacterial communities associated with sediment and leaf material were not detected. However, tebuconazole exposure did significantly reduce conidia production and altered fungal community composition associated with leaf material. An effect on a higher trophic level was observed when Gammarus pulex were fed tebuconazole-exposed leaves, which caused a significant decrease in their feeding rate. Therefore, tebuconazole may affect aquatic fungi and fungally mediated processes even when applied at its “non-microbial” HC5 concentration.