The parafluvial hyporheic zone combines the heightened biogeochemical and microbial interactions indicative of a hyporheic region with direct atmospheric/terrestrial inputs and the effects of wet/dry cycles. Therefore, understanding biogeochemical cycling and microbial interactions in this ecotone is fundamental to understanding carbon cycling at the aquatic–terrestrial interface and to creating robust hydrobiogeochemical models. We aimed to ( i ) characterize biogeochemical and microbial differences in the parafluvial hyporheic zone across a small spatial domain (6 lateral meters) that spans a breadth of inundation histories and ( ii ) examine how parafluvial hyporheic sediments respond to laboratory-simulated reinundation. Surface sediment for assays and forced inundation laboratory incubations (destructively sampled at 0.5 hours and 25 hours) was collected at four elevations along transects perpendicular to flow of the Columbia River, eastern WA, USA. The sampling elevations were inundated by the river 0 days, 13 days, 127 days, and 398 days prior to sampling. Spatial variation in environmental variables (organic matter, moisture, nitrate, glucose, % C, % N) and microbial communities (16S and ITS rRNA gene sequencing, qPCR) were driven by differences in elevation and thus inundation history. Microbial respiration did not differ significantly across elevations prior to inundation. Inundation suppressed microbial respiration relative to uninundated sediment across all elevations, but the degree of suppression was dramatically different between the elevations saturated and unsaturated during sampling, indicating a binary threshold response. We present a conceptual model in which irregular hydrologic fluctuations facilitate microbial communities adapted to local conditions and a relatively high flux of CO 2 . Upon re–wetting, microbial communities are initially suppressed metabolically, which results in lower CO 2 flux rates primarily due to suppression of fungal respiration. Following prolonged inundation, the microbial community adapts via a shift in composition. Our results indicate that the time between inundation events can push the system into alternate states: we suggest that ( i ) above some threshold of inundation–interval, re–inundation suppresses respiration to a consistent, low rate, and ( ii ) that below some inundation–interval, re–inundation has a minor effect on respiration. Extending reactive transport models to capture processes that govern such dynamics will provide more robust predictions of river corridor biogeochemical function under altered surface water flow regimes in both managed and natural watersheds.