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A paleoecological and paleoenvironmental study was conducted on the 2.7 Ga Meentheena Member of the Tumbiana Formation, Fortescue Basin, Western Australia. It involved the integrated application of stratigraphic logging and trace element, stable isotope and organic geochemical analyses. This study aimed to document specific microbial forms and to attribute them to specific stromatolite morphologies, determine the environments they formed in, and search for indications of the presence of molecular oxygen on Earth prior to the Great Oxygenation Event. Twelve stratigraphic sections of the Tumbiana Formation documented stromatolite morphologies given in a proposed sequence of increasing water depth; stratiform, small nodular/bulbous, columnar, pseudocolumnar laminated domical bioherms, stratiform domed biostromes and conically laminated columnar stromatolites. Sedimentary features present (rapid horizontal and vertical facies changes, imbricated intraclasts, symmetrical climbing ripples, and stromatolites of low synoptic relief) indicate a shallow, lacustrine paleoenvironment. The distribution of conically laminated columnar stromatolites suggests they probably formed in a nutrient-poor, deeper water setting, yet within the photic zone. Stable carbon isotope analysis of outcrop samples partially revealed preferential values of lesser 13C depletion in the conically laminated stromatolites (average δ13Corg, -15.90‰), and extreme 13C depletion (average δ13Corg -45.02‰) among the other stromatolite morphologies from the suggested shallower water components. The values do not differ substantially among non-conically laminated stromatolite morphologies, and suggest widespread methane cycling. δ13Ccarb (avg. -0.7‰) values do not correlate with δ13Ocarb (avg. -18.10‰) indicating that there is no diagenetic over-printing and suggest spatially and temporally variable environments. Hydrocarbon compounds including n-alkanes, isoprenoids, monomethylalkanes, hopanes, and steranes were extracted from outcrop samples with no modern contaminants detected. Thermal maturity indicators from steranes and aromatic hydrocarbons are consistent with mineral indicators of thermal history and indicate syngeneity. Hydrocarbon signatures given at different localities and stromatolite morphologies differed and hydrocarbon compounds were shown to increase in concentration towards the centre of a conically laminated columnar stromatolite outcrop sample further suggesting a syngenetic source. Compound ratios suggest a sulphate–poor lacustrine paleoenvironment further supported by trace element analysis of stromatolites which demonstrate a smooth normalised plot. Organisms that undertook bacterial and/or eukaryotic sterol synthesis, consistent with other findings from rocks in this area and of this age, were present in conically laminated columnar stromatolites and suggest at least mild oxygenation at this time in Earth's history, within the deeper sections of a shallow lake.