Gas chromatography–mass spectrometry of oil inclusions from the ca. 1430 Ma marine Roper Group in the Roper Superbasin, Australia, provides a new source of information about the early biosphere and Proterozoic petroleum systems. Oil most likely derived from an overlying shale was trapped at ∼60 °C as abundant oil inclusions within transgranular microfractures in detrital quartz during Mesoproterozoic basin inversion. The oil is very mature and has a wide range of biomarkers, derived mainly from cyanobacteria, but lacks eukaryote biomarkers. Unlike associated solid bitumens, the inclusion oil is nonbiodegraded. Evidently, the inclusions remained closed systems, sheltered from postentrapment alteration and contamination. Because fluid inclusions have preserved biomarkers for >1000 m.y., they constrain the diversity of primordial ecosystems, whereas other forms of early Precambrian organic matter are usually absent or metamorphosed.