We report detailed molecular geochemistry of oil-bearing fluid inclusions from a ca. 2.45 Ga fluvial metaconglomerate of the Matinenda Formation at Elliot Lake, Canada. The oil, most likely derived from the conformably overlying McKim Formation, was trapped in quartz and feldspar during diagenesis and early metamorphism of the host rock, probably before ca. 2.2 Ga. The presence of abundant biomarkers for cyanobacteria and eukaryotes derived from and trapped in rocks deposited before the Great Oxidation Event is consistent with an earlier evolution of oxygenic photosynthesis than previously thought and suggests that some aquatic settings had become sufficiently oxygenated for sterol biosynthesis by this time. It also implies that eukaryotes survived several extreme climatic events, including the Paleoproterozoic “snowball Earth” glaciations. The extraction of biomarker molecules from Paleoproterozoic oil-bearing fluid inclusions thus establishes a new method, using low detection limits and system blank levels, to trace evolution of life through Earth's early history that avoids the potential contamination problems affecting shale-hosted hydrocarbons.