Background. The spatial and temporal changes of assemblages of aquatic insect can be used to detect the anthropic impacts that influence the biological communities. Goals. We compared the assemblages of aquatic insect in 1997 and 2014 in two subtropical river drainages, the association with water characteristics, and we discuss their implications for ecosystems conservation. Methods. True diversity of the aquatic insect fauna at family level and their community structure for 27 study sites in 1997 and 2014 were assessed. Multivariate analyzes were used to compare aquatic insect assemblages and the abundance of functional feeding groups. Results. There were significant differences in the dissolved oxygen (DO) of the water between 1997 and 2014, decreasing its values. Other variables correlated to DO were also modified, with a decrease in pH and an increase in temperature. We found a correlation between reduction of DO and water pH with a decline in the overall abundance of aquatic insects; also, with shifts in the community structure, from the decrease of groups such as some Ephemeroptera and scrapers, to the increase in opportunistic families such as Chironomidae, Culicidae, and other predator families such as Coenagrionidae, Corixidae and Veliidae, and more abundance of collectors. Families such as Heptageniidae and Caenidae decreased in abundance, as well as other benthic groups. Conclusions. The assemblages of aquatic insect are useful to indicate a generalized degradation of environmental conditions across localities and time in two subtropical river drainages, related to water quality degradation symptoms such as reduction of pH levels and dissolved oxygen, usually associated with anthropogenic stressors.