Urban development has put great pressures on lotic environments, as human needs have often been prioritized over ecosystem health. Typical interventions found in urban watercourses include channelization and straightening, clearance of riparian vegetation, and the discharge of wastewaters, among others. Such environmental changes often have major effects on aquatic biodiversity. The main goal of this study was to investigate how freshwater macroinvertebrate assemblages responded to nearly three decades of population growth and infrastructure development in the cities of Esquel and Trevelin (Patagonia, Argentina). Data from two field surveys was used to make comparisons; the first one was conducted in 1990–91, and the second one, in 2017–18. We found significant differences between surveys in several water quality variables, highlighted by an increase in conductivity and a decrease in oxygen availability. Sites affected by channelization presented lower diversity and abundance of mayflies (Ephemeroptera) and stoneflies (Plecoptera), likely due to alteration of the physical habitat and riparian vegetation. Similarly, sites located downstream of wastewater treatment discharges exhibited impoverished communities dominated by stressor-resistant taxa (e.g., Chironomidae). Statistical analyses showed that temporal dissimilarities between sampling sites were best explained by taxa losses, and that these changes were driven by an increase in water conductivity and a decrease in dissolved oxygen. Our findings show a pattern of ecological degradation that is all too common in urban streams. In addition, pressure on lotic ecosystems is only expected to increase due to changes in thermal and precipitation regimes, and increases in human demand for freshwater. Therefore, we provide a series of measures that could contribute to improve stream health and mitigate common urbanization-driven impacts.