Cyanobacteria are able to survive in various extreme environments via the production of organic compounds known as compatible solutes. In particular, cyanobacteria are capable of inhabiting hypersaline environments such as those found in intertidal regions. Cyanobacteria in these environments must possess regulatory mechanisms for surviving the changing osmotic pressure as a result of desiccation, rainfall and tidal fluxes. The objective of this study was to determine the compatible solutes that are accumulated by cyanobacteria from hypersaline regions, and specifically, the stromatolite ecosystems of Shark Bay, Western Australia. Previously, the cyanobacterial populations associated with these stromatolites were characterized in two separate studies. Compatible solutes were extracted from isolated cyanobacteria here and identified by nuclear magnetic resonance. As the media of isolation contained no complex carbon source, the solutes accumulated were likely synthesized by the cyanobacteria. The data indicate that from this one habitat taxonomically distinct cyanobacteria exposed to varying salinities accumulate a range of known compatible solutes. In addition, taxonomically similar cyanobacteria do not necessarily accumulate the same compatible solutes. Glucosylglycerol, a compatible solute unique to marine cyanobacteria was not detected; however, various saccharides, glycine betaine, and trimethylamine-N-oxide were identified as the predominant solutes. We conclude that the cyanobacterial communities from these hypersaline stromatolites are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. The characterization of osmoregulatory properties of stromatolite microorganisms provides further insight into how life can thrive in such extreme environments.