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The “University of Coimbra-Alta and Sofia” area was awarded the UNESCO World Heritage Site distinction in 2013. The Old Cathedral of Coimbra, a 12th-century limestone monument located in this area, has been significantly impacted during the last 800 years by physical, chemical, and biological processes. This led to the significant deterioration of some of its structures and carvings, with loss of aesthetical, cultural, and historical values. For this work, deteriorated spots of the walls of three semi-open chapels from the cloister of the Cathedral were sampled to ascertain their bacterial and archaeal structural diversity. Based on Next-Generation Sequencing (NGS) result analysis, we report the presence of microbial populations that are well adapted to an ecosystem with harsh conditions and that can establish a diverse biofilm in most cases. While it was possible to determine dominant phylogenetic groups in Archaea and Bacteria domains, there was no clear connection between specific core microbiomes and the different deterioration patterns analyzed. The distribution of these archaeal and bacterial communities within the analyzed biodeterioration spots suggests they are more influenced by abiotic factors (i.e., water availability, salinity, etc.), although they influence (and are influenced by) the algal and fungal population composition in this ecosystem. This work provides valuable information that can assist in establishing future guidelines for the preservation and conservation of this kind of historic stone monuments.