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The benthic biolayer is a shallow zone of reactive streambed sediments, widely believed to contribute disproportionately to whole‐stream reactions such as aerobic respiration and contaminant transformation. Quantifying the relative contribution of the biolayer to whole‐stream reactions remains challenging because it requires that hyporheic zone solute transport and reaction heterogeneity are explicitly captured within a single modeling framework. Here, we use field experiments and modeling to quantify the biolayer's aerobic reactivity relative to other stream compartments. We co‐injected and monitored several fluorescent tracers, including the reactive tracer resazurin, into a controlled experimental stream. We characterized reactive transport in the water column and at multiple depths in the hyporheic zone by fitting all data to a new mobile‐immobile model, using resazurin‐to‐resorufin conversion as an indicator of aerobic bioreactivity. Results show that the biolayer converted 8 times more resazurin to resorufin than all other stream compartments, and 80% of this conversion occurred within 2 reach advection times. This hotspot and hot moment behavior is attributed to the biolayer's ability to rapidly acquire, transiently retain, and rapidly degrade stream‐borne solutes. The model analysis shows that the majority of raz‐to‐rru conversion occurs in the biolayer across streams with a wide range of biolayer structural properties, including streams with a biolayer that is less reactive than deeper regions of the hyporheic zone. Together, our results show that the biolayer is a common feature of streams and rivers that should be considered in network‐scale models of aerobic reactivity.