Didymosphenia geminata (Didymo) is a benthic, freshwater diatom that has reached nuisance biomass levels in streams throughout the world. The stalks used to attach cells to substrata can form mats that completely cover stream bottoms and alter benthic community structure; however, it is unclear how Didymo influences energy flow in benthic food webs. We investigated the effects of Didymo mats on community resource use in the Clinch, South Holston, and Watauga rivers in Tennessee. We sampled each river in 2014 and 2015 along a spatial gradient downstream of dams that corresponds to a gradient of mat coverage. We measured stable carbon and nitrogen isotope ratios of major food web components to assess potential changes in macroinvertebrate and fish resource use due to didymo mat proliferation. In 2015, we also used lipid profiles to better isolate the effects of Didymo on resource use. Macroinvertebrate isotopic signatures consistently suggested shifts in resource use in the presence of high (>50%) Didymo coverage from rock biofilms to macrophytes. Brown and rainbow trout in both high and low Didymo sites appeared to primarily be assimilating materials from turbellarians and Didymo stalk material, with less input from amphipods and other macroinvertebrates. Lipid analyses from 2015 supported isotope results. Didymo mats change food resource availability and macroinvertebrate assemblages adapt to using what is available. Trout also appear to consume Didymo mats but do not change what they consume in low and high mat coverage stream reaches