Aim We evaluated the effects of native vegetation and agricultural activities on functional characteristics of aquatic insects’ assemblages in Atlantic Forest streams southern Brazil. Methods We collected information on land uses, riparian zone structural characteristics, and limnological variables of ten streams to characterize their environmental quality. In the same streams, we collected aquatic insects (Ephemeroptera, Plecoptera, and Trichoptera) and determined their functional characteristics (e.g. food habits, mobility, shape, and body size). The information for each trait category was quantified using a fuzzy code technique. To test the environmental conditions on aquatic insect functional attributes, we used an RLQ analysis. Results In our study, only five environmental variables showed variation in streams (temperature, pH, DO, native vegetation, and agriculture use). We collected a total of 2591 organisms distributed in 21 EPT genera. We observed that the functional attributes of EPT formed distinct groups in relation to the stream categories defined by the measured environmental variables. We observed that shredders and larger and flattened organisms were associated with natural streams. These attributes were associated with the genera Phylloicus, Anacroneuria, Tupiperla, and Farrodes. On the other hand, we observed greater proportions of scrapers, with spherical bodies and shelter builders in streams impacted by agriculture. The most frequent genera in these streams were Itaura, Wormaldia and Helicopsyche. Conclusions The modification of natural landscapes by agricultural areas caused significant functional changes in the aquatic invertebrate communities. Since aquatic insects participate effectively in ecological processes, alterations in the functional characteristics of these communities can cause changes in the streams’ functioning.