The benthic biolayer is a reaction hotspot that contributes disproportionately to whole-stream reactions, including aerobic respiration and contaminant transformation. Quantifying the relative contribution of the biolayer to whole-stream mass transformation remains challenging because it requires that hyporheic zone solute transport and reaction heterogeneity are explicitly captured within a single upscaled modeling framework. Here, we use field experiments and modeling to quantify mass transformation in the biolayer relative to other stream compartments. We co-injected and monitored several fluorescent tracers, including the reactive tracer resazurin, into a controlled experimental stream whose 8.5 cm hyporheic zone is isolated from groundwater. We characterized reactive transport in the water column and at multiple hyporheic zone depths by simultaneously fitting concentration time series measured at these locations to a new mobile-immobile model, using resazurin-to-resorufin transformation as an indicator of aerobic bioreactivity. Results show that the biolayer transformed 2× more resazurin to resorufin than all other stream compartments combined, and over half of all reactions occurred within 2 advective travel times through the reach. This hotspot and hot moment behavior is attributed to the biolayer’s propensity to rapidly acquire, transiently retain, and rapidly degrade stream-borne solutes. Model analysis shows that mass transformation is highest in the biolayer across a wide range of biolayer structural properties, including scenarios when the biolayer is less reactive than deeper regions of the hyporheic zone. Together, our results point to the biolayer as a common feature of streams and rivers that should be considered in network-scale models of river corridor biogeochemistry.