Little research has been conducted on the influence of microbial communities, sediment disturbances, and their interaction on the Vertical Water Flux (VWF) across a hydraulically complex streambed. Our study was aimed at the effects of microbial activity and shallow-sediment disturbance on VWF in sandbed flumes. We assessed the dynamics of VWF and the development of a microbial community during 30 d (June–July 2010) in 16 outdoor flumes with 2 types of bedform shape (level and ripple). We operated 8 flumes in constant darkness (no-light), and 8 in daylight to gain information on the relative significance of algae and heterotrophic microorganisms. We simulated a shallow-sediment disturbance after 21 d and compared microbial activity and VWF before and after the disturbance. We measured organic matter content, abundance of organisms, microbial activity, precipitation of CaCO₃, and O₂ bubbles resulting from primary production before and after the disturbance. VWF differed among treatments after 13 d. Algal and bacterial cells embedded in an extracellular polymer network, algal and microbial precipitation of CaCO₃, and production of O₂ bubbles in the uppermost sediment blocked the sediment pore space and disrupted VWF under daylight conditions, whereas bacterial cells and microbial precipitation of CaCO₃ reduced VWF in no-light flumes. Microbial activity and organic matter were not affected by the shallow-sediment disturbance, but VWF was restored. VWF can be controlled by microbial activity and shallow-sediment disturbances and should be seen as spatially heterogeneous and fluctuating in time on small scales.