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Abstract Microbial mats are commonly observed in estuaries and in salt marshes but they only rarely represent a significant surface involved in salt production. In the Sečovlje salt works in Northern Adriatic, a microbial mat known as the “petola” covers the bottom of salt crystallising pans, highly influencing salt composition and salt production processes. Throughout the year the petola is subjected to numerous co-varying factors that drive changes in its structure and the microbial community. Seasonal modifications were investigated via various methods (cryo-HRSEM, XRD, elemental analysis, carbohydrate content, bacterial community structure). This study provides knowledge on microbial mat compositional characteristics and functional roles in response to seasonal variation in environmental conditions. The in situ characterisation (close-to its natural hydrated state) of the three-dimensional microstructure provides precise information about dominating filamentous cyanobacterium Coleofasciculus chthonoplastes and extracellular polymer secretion (EPS) organisation. This is the first study to address how microbial mat composition and structure, especially 3D EPS network (and microbial diversity), affects the salt production processes within a hypersaline environment. Graphical abstract Unlabelled Image Highlights • Role of a microbial mat called “petola” in traditional Slovenian salt production. • Combination of microscopic, mineralogical, geochemical and biomolecular analyses. • High resolution cryo-scanning electron microscopy. • Petola's 3D organic microtexture impacts salt formation and production. • Magnesium sequestration by cyanobacterial sheaths favours salt purity. [ABSTRACT FROM AUTHOR]