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  • The ca. 1.4 billion years (Ga) old Roper Group of the McArthur Basin, northern Australia, is one of the most extensive Proterozoic hydrocarbon-bearing basins deposited in a large epeiric sea known as the Roper Seaway. Black shales from the Velkerri Formation were deposited in a deep water shoaling sequence and are well preserved in the Altree 2 drillcore in the Beetaloo Sub-basin. These shales were analysed to determine their organic geochemical (biomarker) signatures which were used to interpret the microbial diversity and palaeoenvironment of the Roper Seaway. The results were integrated with published inorganic geochemistry and microfossil distributions. The indigenous hydrocarbon biomarker assemblages describe a water column dominated by bacteria with large scale heterotrophic reworking of the organic matter in the water column or bottom sediment. Evidence for microbial reworking includes a large unresolved complex mixture (UCM) and high ratios of monomethyl alkanes relative to n-alkanes—features characteristic of indigenous Proterozoic bitumen. Steranes, biomarkers for single-celled and multicellular eukaryotes, were below detection limits in all extracts analysed, despite eukaryotic microfossils having been previously identified in the Roper Group. These data suggest that eukaryotes, while present in the Roper Seaway, were ecologically restricted and contributed little to the net biomass. The combination of increased dibenzothiophene in the middle Velkerri Formation and low concentrations of 2,3,6-trimethyl aryl isoprenoids throughout the Velkerri Formation suggest that the water column at the time of deposition was transiently euxinic. As a comparison we reanalysed extracts from the 1.64 Ga Barney Creek Formation of the McArthur Basin. The biomarker assemblages differ between the Velkerri and Barney Creek Formations between is a biomarkers and water column chemistry, demonstrating that the microbial environments and water column geochemistry were variable in the Proterozoic.