Sediment-borne Escherichia coli can elevate waterborne concentrations through sediment resuspension or hyporheic exchange. This study sought to correlate hydrological, sediment transport, and water quality variables with: (i) the temporal stability of sediment E. coli populations [concentrations, strain richness and similarity (Raup–Crick index)]; and (ii) the contribution of sediment E. coli to the water column as defined through a library-dependent microbial source tracking approach that matched waterborne E. coli isolates to sediment E. coli populations. Three monitoring locations differing in their hydrological characteristics and adjacent upland fecal sources (dairy operation, low-density residential, and tile-drained cultivated field) were investigated. Sediment E. coli population turnover was influenced by sediment transport at upstream, high-energy reaches, but not at the downstream low-energy reach. Sediment contributions to the water column averaged 13% and 18%, and fecal sources averaged 17% and 21% at the upstream sites adjacent to dairy operations and low-density residential areas, respectively. Waterborne E. coli at the downstream site had low matches to E. coli from reach sediments (1%), higher matches to the upstream sediments (27% and 12%), and an average of 14% matches to the tile drained field. The percentage of waterborne E. coli matching sediment-borne E. coli at each stream reach varied in correlations to hydrological and sediment transport variables, suggesting reach-specific differences in the role of sediment resuspension and hyporheic exchange on E. coli transport.