Riverbeds are hotspots for microbially-mediated reactions that exhibit pronounced variability in space and time. It is challenging to resolve biogeochemical mechanisms in natural riverbeds, as uncontrolled settings complicate data collection and interpretation. To overcome these challenges, laboratory flumes are often used as proxies for natural riverbed systems. Flumes capture spatiotemporal variability and thus allow for controlled investigations of riverbed biogeochemistry. These investigations implicitly rely on the assumption that the flume microbiome is similar to the microbiome of natural riverbeds. However, this assumption has not been tested and it is unknown how the microbiome of a flume compares to natural aquatic settings, including riverbeds. To evaluate the fundamental assumption that a flume hosts a microbiome similar to natural riverbed systems, we used 16s rRNA gene sequencing and publicly available data to compare the sediment microbiome of a single large laboratory flume to a wide variety of natural ecosystems including lake and marine sediments, river, lake, hyporheic, soil, and marine water, and bank and wetland soils. Richness and Shannon diversity metrics, analyses of variance, Bray-Curtis dissimilarity, and analysis of the common microbiomes between flume and river sediment all indicated that the flume microbiome more closely resembled natural riverbed sediments than other ecosystems, supporting the use of flume experiments for investigating natural microbially-mediated biogeochemical processes in riverbeds.