We here show that nano-scale mapping of elements commonly utilized in biological cycles provides a promising new additional line of evidence when evaluating the extent of the contribution of biology to microbialites. Our case study comes from Lake Clifton in Western Australia, a unique environment where living domical and conical microbialites occur in close proximity to ≤4000-year-old fossilized equivalents. The outer margins of a partially lithified, actively growing Lake Clifton microbialite are characterized by abundant filamentous cyanobacteria within a loosely cemented aragonite matrix. Nano-scale chemical maps have been successfully matched to specific morphological features such as trichomes, sheaths and putative extracellular polymeric substances (EPS). A suite of elements (C, O, Mg, N, Si, S) is concentrated within cyanobacterial sheaths, with carbon, magnesium, nitrogen and sulfur also enriched within trichomes and putative EPS. Calcium distribution highlights the sites of aragonite mineralization. In contrast, the fossilized Lake Clifton microbialite contains only rare, extensively degraded cyanobacterial filaments, the mean diameter of which is <50% of the living equivalents. Nevertheless, nano-scale chemical maps can again be matched with morphological features. Here, poorly preserved filamentous microfossils are highlighted by enrichments in nitrogen and sulfur. Magnesium is no longer concentrated within the filaments, instead it co-occurs with calcium and oxygen in the calcite cement. Extension of this study to a ~2720-million-year-old stromatolitic microbialite from the Tumbiana Formation of Western Australia shows that similar nano-scale signals, in particular nitrogen and sulfur enrichments, are characteristic of stromatolite laminations, even when morphological microfossils are absent. The close similarities of nano-scale elemental distributions in organic material from modern and ancient microbialites show that this technique provides a valuable addition to the morphological investigation of such structures, particularly in non-fossiliferous ancient examples.