The enigmatic Paleozoic fossil PrototaxitesDawson 1859 consists of tree-like trunks as long as 8 m constructed of interwoven tubes <50 mm in diameter. Prototaxites specimens from five localities differ from contemporaneous vascular plants by exhibiting a carbon isotopic range, within and between localities, of as much as 13‰ δ13C. Pyrolysis–gas chromatography–mass spectrometry highlights compositional differences between Prototaxites and co-occurring plant fossils and supports interpretation of isotopic distinctions as biological rather than diagenetic in origin. Such a large isotopic range is difficult to reconcile with an autotrophic metabolism, suggesting instead that, consistent with anatomy-based interpretation as a fungus, Prototaxites was a heterotroph that lived on isotopically heterogeneous substrates. Light isotopic values of Prototaxites approximate those of vascular plants from the same localities; in contrast, heavy extremes seen in the Lower Devonian appear to reflect consumption of primary producers with carbon-concentrating mechanisms, such as cryptobiotic soil crusts, or possibly bryophytes. Prototaxites biogeochemistry thus suggests that a biologically heterogeneous mosaic of primary producers characterized land surfaces well into the vascular plant era.