Exchange between groundwater (GW), hyporheic zone waters (HZ) and surface waters (SW) is critical for water quality, quantity, and the ecological health and functioning of all three ecosystems. Hydrological exchange is particularly important in intermittent creeks, such as in the Murray Darling Basin, Australia, where stream reaches shift from losing to gaining depending on the volume of surface flows. In this study we used hydrochemistry to identify SW-GW exchange and combined this with eDNA data to analyse the response of eukaryote and prokaryote communities to differing flow conditions within intermittent and perennial stream reaches. Our study suggested that SW and GW microbial communities were only around 30% similar. Differences in microbiota between SW, HZ and GW habitats were driven by changes in relative abundances of surface water dominant organisms (such as those capable of photosynthesis) as well as anaerobic taxa typical of GW environments (e.g., methanogens), with GW and HZ microbial communities becoming increasingly different to those in SW as flow ceased in intermittent creeks. Fine-scale hydrologic changes were identified through microbial communities in the perennial Maules Creek, indicating the importance of GW-SW exchange to biotic communities. This study highlights the importance of flow in shaping microbial communities and biogeochemical cycling within intermittent creeks and their connected alluvial aquifers. Our results suggest that microbiota may prove a useful indicator of SW-GW exchange, and in some circumstances, may be more sensitive in demonstrating fine-scale changes in SW-GW interactions than water chemistry. This knowledge furthers our understanding of GW-SW exchange and its impacts on ecological health.