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Microorganisms, including fungi, bacteria, cyanobacteria, and algae, frequently colonize the surfaces of cultural heritage materials. These biological agents can cause biodeterioration through various mechanisms, resulting in aesthetic alteration, physical disruption, or compromise of mechanical integrity. To assess the presence and diversity of microorganisms, a combination of microscopy techniques is commonly used in conjunction with results from both culture‐dependent and culture‐independent methods. However, microbial populations are often underestimated. To address this issue, microorganisms can be detected by their intrinsic fluorescence, which can be observed via fluorescence microscopy. This approach facilitates the mapping of the spatial arrangement of microorganisms and the understanding of colonization patterns, thereby complementing established imaging techniques and providing insight into the interactions of microbial communities with the substrate. Given the limited research in this area, we examine the potential of microorganism autofluorescence as a molecular tool for investigating biodeterioration in artistic and architectural heritage, with a particular focus on paper and stone materials. Identifying and understanding the diverse microbiota that may be present is crucial for developing tailored and effective preventive measures and conservation treatments, as some of the species discovered may pose significant risks to both artifacts and human health. [ABSTRACT FROM AUTHOR]