Raman spectra of carbonaceous materials in one of the world's oldest sedimentary rock formations – the Strelley Pool Chert (Pilbara Craton, Western Australia) – are analysed to determine whether primary structural characteristics of organic molecules may have survived to the present day. We use Raman spectral parameters to identify variations in molecular structure of the carbon and determine whether original characteristics of the carbonaceous materials have been completely thermally overprinted, as would be expected during c.3.5 billion years of geologic history. To the contrary, we find that the molecular structure of the carbonaceous materials varies depending on the sedimentary layer from which the sample came and the inferred original palaeoenvironmental setting of that layer, as determined by other geochemical and geological data. Thus, we argue that the spectral characteristics of the carbonaceous materials reflect original palaeoenvironments that varied through time from warm hydrothermal settings to cooler marine conditions and a return to hydrothermal conditions. Raman spectroscopy is also used to show that organic matter is present in trace amounts in association with putative stromatolites (laminated sedimentary structures possibly formed by microorganisms) in the Strelley Pool Chert, which were previously thought to be devoid of organic remains. Furthermore, the Raman spectra of carbon associated with stromatolites indicate lower thermal maturity compared to the carbon in (non-stromatolitic) hydrothermal deposits above (younger) and below (older). Significantly, this indicates that the stromatolites are not abiotic hydrothermal precipitates – as previously proposed – but were formed in a cooler marine environment that may have been more favorable to life.