Biological aerosols from air constitute a significant source of exposure to microorganisms in public places. Airborne microorganisms are involved in the development of certain respiratory symptoms, allergies, or infections among users and occupants. Various sampling instruments have commonly been used in aerobiology to collect bacteria and fungi suspended in the air. The objective of this study was to develop a reliable procedure for sampling in indoor public environments presenting different levels of occupancy, airborne bacteria and fungi to be subjected to molecular analysis (bacteria and fungi quantitative PCR, capillary electrophoresis single strand conformation polymorphism fingerprinting). Four different sampling devices were tested in situ in an office building (open-plan type) and the sampling strategy chosen was tested in two museum contexts. In accordance with the drawbacks involved to our study (quantitative and qualitative aspects, cost, and overcrowding), cyclone device appeared to be most suitable. The results underline the effectiveness of this high-volume aerosol sampling device for both qualitative and quantitative molecular analysis. Four in situ sampling collections were carried out in 1 day in the Louvre Museum to study quantitative and qualitative variations of airborne bacterial and fungal diversity. The quantitative results revealed a similar order of magnitude for the numbers of both bacteria and fungi. In the Louvre Museum, the samples yielded between 3.7x104 and 4.1x104 genome equivalent (GE) bacteria/m3 air and between 5.0x104 and 5.9x104 GE fungi/m3 air and in the Decorative Arts Museum between, 2.1x104 and 2.5x104 GE bacteria/m3 air and between 1.4x104 and 1.7x104 GE fungi/m3 air. The results also indicate that the dominant bacterial community displayed a stable structure over a short period of time whereas dominant eukaryotic airborne community appeared more variable.