Between 2014 and 2019, Central Europe was subject to unusually high temperatures and periods of drought due to climate change, with a subsequent occurrence of low and zero flows. As aquatic organisms have shaped their life strategies in direct relation to natural hydrological regimes, any changes to these regimes necessarily lead to a cascade of responses in biota species composition in the ecosystem concerned. Here, we examine changes in aquatic macroinvertebrates assemblage composition and diversity in a small fishless headwater brook in a highland area of the Czech Republic that took place between 2010 and 2019. While flow rates were relatively constant prior to 2014, the hydrological regime was significantly influenced by periods of severe drought between 2017 and 2019, with periods of zero discharge and dry riverbed. The shift from a perennial to an intermittent stream led to an increase in generalist taxa abundance and a decline in lotic taxa, while the abundance of lentic and semi-aquatic taxa was subjected to fluctuations non-related to the hydrological regime (drought occurrence). During the period of droughts, the abundance of taxa recognised as indicators of good quality water declined, while the numbers of those indicating moderately to heavily polluted environments increased.