Carbon and nitrogen stable isotopic compositions of fossil materials from Lake Messel (47 Ma) in Germany are used to investigate Eocene ecosystem dynamics. Autolithified soft tissues of terrestrial and aquatic vertebrate organisms, as well as plant compression fossils, contain organic material (20–50 wt% C, 1–6 wt% N), which appears to retain precursor compositions. Stable isotopic compositions (δ13C and δ15N) of Messel fossils are similar to those reported for components in modern lacustrine ecosystems. These data show trophically sensible enrichments relative to food sources, reflect multiple feeding strategies for each organism (e.g., omnivory, planctivory, piscivory), and differentiate between benthic and pelagic organic carbon sources. These chemical data broadly confirm existing Messel food web models based on coprolite and gut content analyses. δ13C values for the lacustrine shale range from −30.3 to −26.3‰, pointing to mixed terrestrial and aquatic origins for primary producers in the food web. δ13C values for primary consumers such as insects overlap with those for primary producers but are comparatively enriched in 15N. Secondary and higher consumers (fish, crocodiles, and frogs) are associated with even more positive δ15N values and show a more constrained range of δ13C values. Omnivory appears widespread in both low and high trophic level consumers. Hence, the stable isotopic compositions of Messel fossils are complex and overlap, and must be combined with paleontological investigations in order to be conclusive. This study represents the first comprehensive isotopic reconstruction, featuring tens of components, of an ecosystem of Eocene age. A thorough understanding of trophic structure in Eocene Lake Messel contributes to the global databank of ecological history.