Changes in the concentration of microbial aerosol were examined in the selected premises of the Jagiellonian University Museum in Kraków, made available to visitors during the Night of Museums, i.e. Hall, Libraria and Treasury. The samples were collected four times using the collision method with MAS-100 air sampler. The concentration of mesophilic and heamolytic bacteria, molds, staphylococci and actinomycetes was examined and an attempt was made to find the correlation between the number of museum visitors and the concentration of microbial aerosol. The smallest number of visitors (600 persons) was recorded in Libraria, while the greatest (900 persons) in the Hall. The prevalence of microorganisms varied significantly between the hours of sampling, from the smallest number in the morning, after opening, to the greatest recorded while there were most visitors (9 p.m.). The largest number of molds was observed after the museum was closed (0:30). A strong positive correlation was found between the concentration of airborne mesophilic bacteria and actinomycetes with the number of visitors, the concentration of fungi was negatively correlated and there was a weak positive correlation in the case of staphylococci. On average, the greatest concentration of mesophilic bacteria (860 CFU/m3), actinomycetes (60 CFU/m3) and staphylococci (26 CFU/m3) was recorded in the Hall at 9 p.m. The largest concentration of molds (1,290 CFU/m3) was found in the Hall and Treasury at 0:30. The observed concentrations of airborne microorganisms do not exceed acceptable levels for public utility premises, therefore they do not pose a threat to the health of the Museum employees, tourists and to the condition of the Museum collections.