A summer study of the vertical structure of bi-layered microbial mats was carried out on Byers Peninsula (Livingston Island, South Shetland Islands). These benthic communities had a common basic structure that consisted of two distinct layers differing in composition, morphology and colour. Our sampling focused on mats showing more layering, which thrived over moist soils and at the bottom of ponds. The photosynthetic pigments analysis performed by high-performance liquid chromatography demonstrated a major occurrence of cyanobacteria and diatoms on these mats, the former being more abundant in relative terms on the surface and composed by morphospecies grouping into orders Oscillatoriales, Nostocales and Chroococcales. The areal chlorophyll a concentrations were slightly higher in the deeper layer although not significantly. Our microscopic and chemical analyses showed that non-active biomass accumulates at the surface. Hence, the upper layers showed the sheath pigment scytonemin and higher amounts of exopolysaccharides, as a strategy to cope with environmental stress. On the other hand, the basal layer was composed of more active photosynthetic microbiota, which also revealed a more balanced stoichiometry. Here we exemplify how environmental stresses are potentially overcome by physiological mechanisms developed by microbial mats which also shape their vertical structure.