Groundwater ecosystems are dependent on the input of oxygen, nutrients and organic matter from the surface. The hyporheic zone, the ecotone that connects surface and groundwaters, is a vital conduit for the supply of these nutrients to aquifers. The dynamics of exchange through this zone has a major influence on the biota and the overall health of both surface and groundwater ecosystem, particularly during low flow or drought, when groundwater discharged to streams maintains baseflow. However, surface -groundwater interactions are equally important to the ecosystems within aquifers during losing conditions. This study investigates how variations in surface-groundwater exchanges and connectivity influence the biotic communities in these freshwater ecosystems, using a combination of environmental DNA (eDNA) to characterise microbial and invertebrate communities and water chemistry (including nutrient, carbon and isotope data) to quantify the extent of SW-GW exchange. Samples were collected between 2015-2016 at three locations within the Maules Creek catchment, a sub catchment of the Murray-Darling Basin in NW NSW. At each site, samples were collected along a gradient from the creek through the hyporheic zone and into the adjacent alluvial aquifer. We found a rich diversity of invertebrates and microbes with distinct overlaps in biological communities noted along our gradients. Surface water microbes, distinguished by their ability to photosynthesise, were observed in highly connected aquifers, supporting the direction of water flow indicated by water chemistry data. Our results help characterise biogeochemical processes within the hyporheic zone, highlighting the importance of this hydrological connectivity for surface and groundwater ecosystems. Such findings will lead to a greater understanding of the connectedness of ground- and surface waters resulting in a more holistic view to water management within the Murray-Darling Basin, and elsewhere.