Large‐scale and intensive human activities have caused widespread and profound changes in the diversity and structure of biological communities. Historical or long‐term data are an important tool for the quantification and understanding of temporal changes. A comparison of Ephemeroptera collected at 60 streams in the Morava and Odra river basins in two periods, 1955–1960 and 2006–2011, enabled an estimate of changes in their assemblages induced by various human pressures to be made. The taxonomic composition of mayfly assemblages was substantially dissimilar between the two periods, although the changes were not associated with a decline in regional or local diversity. Only lowland rivers lost several of their original species, mostly habitat specialists. Species replacement, a leading driver of dissimilarity, caused shifts towards more simplified, less specialized assemblages in large rivers, and towards more pollution‐ and siltation‐tolerant assemblages in small rivers. The increase in cold‐water specialists and a stable share of generalists suggested the maintenance of a certain level of specialization in the assemblages of brooks. The most marked change in the assemblage was associated with the impaired or bad water quality of rivers in the 1950s, which persisted or further deteriorated in the ensuing decades. Assemblages that were influenced by a slight deterioration or improvement in water quality were less altered, unless affected by other pressures (such as channel or discharge modifications). The results indicate that the causes of changes in the assemblages are many and complex, although heavy pollution overrides other influences. Direct habitat loss or their degradation by siltation appear to be leading contributors to changes in assemblages. The results imply the need for the application of a catchment‐scale perspective for the restoration of streams and the conservation of the remaining well‐preserved stretches of lowland rivers. Copyright © 2014 John Wiley & Sons, Ltd.