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A longstanding challenge in stream ecology is to understand how landscape configuration organizes spatial patterns of ecosystem function via lateral groundwater connections. We combined laboratory bioassays and field additions of a metabolic tracer (resazurin) to test how groundwater-stream confluences, or “discrete riparian inflow points” (DRIPs), regulate heterotrophic microbial activity along a boreal stream. We hypothesized that DRIPs shape spatial patterns and rates of aquatic heterotrophic microbial activity by supplying labile dissolved organic matter (DOM) to streams. Laboratory bioassays showed that the potential influence of DRIPs on heterotrophic activity varied spatially and temporally, and was related to their DOM content and composition. At the reach scale, DRIP-stream confluences elevated the spatial heterogeneity and whole-reach rates of heterotrophic activity, especially during periods of high land–water hydrological connectivity. Collectively, our results show how the arrangement of lateral groundwater connections influence heterotrophic activity in streams with implications for watershed biogeochemical cycles.