This dataset includes stable nitrogen-15 and carbon-13 isotope analysis data derived from freshwater aquatic and semi-aquatic primary producers, invertebrates, and fishes collected from five sampling sites (and one pilot site) within the Owens River Gorge, Mono and Inyo counties, CA, USA between 2017 and 2021. These sampling efforts were conducted in conjunction with the California Department of Fish and Wildlife’s fish population surveys and invertebrate monitoring. Fishes sampled were Owens Sucker (Catostomus fumeiventris (Miller, 1973)), hybrid Tui Chub (Siphateles bicolor snyderi (Miller, 1973) x Siphateles bicolor obesa (Girard 1856)), Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) and Brown Trout (Salmo trutta (Linnaeus, 1758)). Invertebrates sampled included 33 taxa and primary producers sampled consisted predominantly of stems and leaves of vascular macrophytes and non-vascular primary producers consisting of 22 taxa. This dataset includes all organism metadata (Dissection_Table.csv, Eye_Table.csv) and N15 and C13 stable isotope analysis data (Isotope_Table.csv) derived from the invertebrates, vegetation, and a representative subset of the fishes collected in this study. These data were collected to assess the effects of restoration flows on aquatic food webs and fish communities in the Owens River Gorge.