Cultural heritage objects provide valuable historical information, but can also harbour biological threats. Still, little is said about the potential risks that may await unaware researchers, conservators, and archaeologists. Our work discusses the study results from the crypts in Krakow, which were opened for the first time. The human and coffin remains were examined. The number of actinomycetes, other mesophilic bacteria, bacterial spores, and xerophilic and non-xerophilic fungi was determined. In general, a low number of microbes was observed. However, scanning electron microscope (SEM) images showed many bacterial conglomerates and confirmed that microbial activity affected the fibres covering cadavers in the crypts. The most abundant were mesophilic bacteria, followed by bacterial spores and actinomycetes. They reached up to 107 CFU/g in fabric remains, 5.2 × 106 CFU/g in burial remains, and 1.6 × 106 CFU/g found under the coffin, and above 7.5 × 105 CFU/g for xerophilic and non-xerophilic fungi. NGS (Next-Generation Sequencing) results suggested that the low presence of microorganisms may be due to the dominance of unculturable or long-growing bacteria belonging to Mycobacterium, such as M. coloregonium, M. arupense, and M. pinnipedii. Moreover, other obligatory/non-obligatory pathogens, Bacteroides fragilis, Clostridium botulinum, Coxiella burnetii, Clostridium tetani, Corynebacterium diphtheriae, Enterobacter cloacae, Escherichia coli, Legionella pneumophila, Mycobacterium leprae, Rhodococcus equi, and Staphylococcus aureus have been recorded in examined samples, with the dominance in bone samples. Results indicate the risk of dangerous pathogens present in historical objects, the impact on health may be severe, and the need to use personal protective equipment and proper measures to control the physical conditions of crypts.