The 2.72Ga Tumbiana Formation is a succession of clastic and carbonate rocks outcropping along the southern margin of the Pilbara Craton in Western Australia. It hosts abundant, diverse and exceptionally well-preserved stromatolites and has provided the setting for numerous investigations focussing on the Archaean biosphere. Despite its palaeobiological significance, the overall depositional setting of the Tumbiana Formation remains unclear. Here we present the results of stratigraphic, sedimentological and geochemical investigation of the Tumbiana Formation in the well-known Redmont/"Knossos" area and at several localities in the northwestern Pilbara sub-basin. We suggest these data are best explained by deposition in fluvial and lacustrine environments of an inward-draining continental basin. δ13Corg values vary from -49.9‰ to -15.0‰. Conical stromatolite morphologies, commonly attributed to cyanobacteria, are anomalously little depleted in 13Corg, implying a higher relative contribution of organic matter from phototrophic versus methane cycling metabolisms.