The present study aims to determine the prevalence of bio‒pollutants in the air at different locations inside Prince Mohammad Ali's museum, in relation to microenvironmental conditions, location characteristics, ventilation type, and human activity. Samples of Airborne bacteria, fungi and actinomycetes were collected using Andersen 2 stages sampler and particles were separated into fine (<2.5 µm) and coarse (>7µm) size ranges. Annually indoor concentrations of bacteria, fungi and actinomycetes ranged from 694–7787 CFU/m³, 47–1985 CFU/m³ and 0–294 CFU/m³, respectively. Seasonally, the highest concentrations of bacteria, fungi and actinomycetes were found in spring, winter and autumn seasons, respectively. Indoor microbial fine fraction (< 2.5 μm) constituted ~ 69–76.5%, 71.5–92% and 65.6–95% for airborne bacteria, fungi and actinomycetes, respectively. Indoor/ outdoor ratios (I/O) exceeded 1 in the reception hall for bacteria, in the residence hall for fungi and in both of the reception hall and restoration laboratory for actinomycetes. The Indoor Global Index of Microbial Contamination (GIMC–CFU/m³) exceeded the limit value of 1000 CFU/m³ in the all sites inside the museum. Naturally ventilated locations had the worst indoor microbial quality. Temperature detrimentally affected microbial culture ability, and relative humidity comparatively supported their culture ability, without clear correlation pattern.