Freshwater ecosystems are endangered worldwide by various human pressures, resulting in dramatic habitat and species loss. Many aquatic invertebrates respond to disturbances in their habitat, and mayflies are among the most sensitive ones. Therefore, we investigated mayfly response to anthropogenic disturbances at 46 study sites encompassing slightly to heavily modified small and mid-sized lowland streams and rivers. Mayfly nymphs were sampled between April and September 2016 using a benthos hand net. A total of 21 species was recorded, with Cloeon dipterum (Linnaeus, 1761) being the most frequently recorded one. Nevertheless, the taxa richness was rather low per site, i.e., between zero and nine. Assemblage structure had a high share of lower reaches and lentic (potamic and littoral) elements, and detritivores (gatherers/collectors and active filter feeders). This indicates that hydromorphological alterations lead to assemblage “potamisation” in small and mid-sized rivers. More mayfly species were related to higher oxygen concentration and lower water temperature, abundance of aquatic vegetation and total organic carbon. Additionally, the assemblage diversity and abundance were negatively associated with increasing intensive agriculture area at the catchment scale. This study confirms mayfly bio-indicative properties, i.e., their sensitivity to alterations of their habitat and pollution, but also provides new data related to mayfly response to the impacted environment. Those data can be used for management and protection activities of lowland rivers and their biota according to the requirements of the European Water Framework Directive.