While many studies focus on either the hyporheic zone or aquifers, no previous studies have sought to compare these two different ecosystems within the same study. This research aims to compare microbial activity in both the hyporheic zone and boreholes. For two 4-6 week sampling campaigns (Spring and Autumn) at Maules Creek, Namoi, New South Wales, Australia, microbial activity was measured using the cotton strip degradation method in the hyporheic zone and adjacent boreholes. In the hyporheic zone, unprimed cotton canvas was affixed to rulers which were then placed in different habitats (dry bar, riffle and pools) at three different water regimes found at different sections of the creek (perennial-gaining, intermittent, and perennial-losing). Cotton strips were also deployed in 13 boreholes located adjacent to the creek. Microbial activity was calculated based on the loss of cotton strip tensile strength. The results of the study showed that microbial activity in the hyporheic zone was greater and slightly more variable compared to the more stable borehole environment. Microbial activity in the hyporheic zone was strongly correlated with the moisture-status of the site, measured during deployment and collection of the cotton strips. These findings may have implications for environmental impact assessments. Sampling for the purposes of environmental impact assessment is often limited to borehole sampling as it remains a simple and cost effective method, while the biogeochemical conditions and ecology of the hyporheic zone have been largely ignored. Incorporating hyporheic zone studies into environmental impact statements may provide a more comprehensive understanding of the ecosystem impacts of agricultural and mining activities.