The observation of oil inclusions trapped prior to 2.0 Ga in Palaeoproterozoic rocks and the ability to obtain detailed molecular geochemical information from them provide a robust way for understanding the early biogeochemical evolution of the Earth. Oil-bearing fluid inclusions (FI) in ca. 2.45 Ga fluvial metaconglomerate of the Matinenda Formation at Elliot Lake, Canada were trapped in quartz and feld-spar during diagenesis and early metamorphism of the host rock, probably before ca. 2.2 Ga. The 2.1 Ga FA Formation sandstone of the Franceville Basin in Gabon that hosts the Oklo natural fission reactors has also been discovered to contain abundant Palaeoproterozoic oil-bearing FIs. This oil occurs within H2O and CO2-dominated inclusions trapped in syntaxial quartz overgrowths and intragranular and transgranular microfractures in detrital quartz, and was most likely trapped 2.1–1.98 Ga. Molecular geochemical analyses of both FI oils reveal a wide range of compounds, including n-alkanes, isoprenoids, monomethylalkanes, aromatic hydrocarbons, and trace amounts of complex multi-ring biomarkers including terpanes, hopanes, methylhopanes, steranes and diasteranes. To ensure a reliable interpretation of oil inclusions, a comprehensive series of outside-rinse blanks and procedural system blanks was analysed by gas chromatography-mass spectrometry; quantitative amounts of the hydrocarbons in these blanks were compared to the FI extracts, so as to provide confidence limits on the experimental integrity of each compound class. Maturity ratios based on reliably detected compound classes show that the FI oils were generated in the oil window, with no evidence of extensive thermal cracking. The presence of biomarkers for cyanobacteria and eukaryotes derived from and trapped in rocks deposited prior to 2.0 Ga is consistent with early evolution of oxygenic photosynthesis and suggests that some aquatic settings had become sufficiently oxygenated for sterol biosynthesis by this time. The extraction of biomarker molecules from Palaeoproterozoic oil-bearing FIs thus establishes a new method, using low detection limits and system blank levels, to trace evolution through Earth’s early history that avoids the potential contamination problems affecting shale-hosted hydrocarbons.