Dolomite [CaMg(CO3)2] is abundant in sedimentary rocks throughout the geological record, but it is rarely found in modern sediments. Also, it cannot be precipitated under low-temperature conditions in the laboratory without microbial mediation and, as a result, its origin remains a long-standing enigma. This study reports biologically mediated dolomite precipitation in ancient microbial mats and biofilms from the Cambrian Tarim Basin. The ambient temperature at the time of dolomite precipitation was estimated from δ18O values from early diagenetic dolomite, and the presence of structures associated with extracellular polymeric substances (EPS), is composed of fibres arranged in a reticular pattern, would favour epitaxial crystallization of dolomite on an organic substrate. In addition, poorly crystallized dolomite formed nanocrystal aggregates that strongly resemble the morphology and size distribution observed in microbial culture experiments. These lines of evidence confirm that microbial structures can be preserved in ancient dolomite and validate their use as biosignatures.