Cultural heritage objects, often aged and vulnerable, are at a particularly high risk of microbial pollution. Enzymatic activity of bacteria and fungi might lead to the damage of precious antique objects, while microbial pathogenicity and immunogenicity can pose a threat to the health of staff and visitors. Therefore, monitoring the microbiological contamination of cultural heritage objects is necessary, especially in the case of unique objects, like tourist trails located underground. We have evaluated the concentration and diversity of microorganisms present in the air and surfaces of the Lublin Underground Trail located in Poland. We have found the prevalence of bacterial species being a part of the human microbiota in the analysed air. The average fungal contamination of the air was low (337 CFU/m³), but we have identified species of high immunogenicity and those contributing to biodeterioration. Microclimatic parameters were considered as favourable for microbial growth, which emphasizes the role of adequate heating and ventilation air conditioning systems providing sufficient air quality in cultural heritage objects.