The microbial content of air has as yet been little described, despite its public health implications, and there remains a lack of environmental microbial data on airborne microflora in enclosed spaces. In this context, the aim of this study was to characterize the diversity and dynamics of airborne microorganisms in the Louvre Museum using high-throughput molecular tools and to underline the microbial signature of indoor air in this human-occupied environment. This microbial community was monitored for 6 month during occupied time. The quantitative results revealed variations in the concentrations of less than one logarithm, with average values of 103 and 104 Escherichia coli/Aspergillus fumigatus genome equivalent per m3 for bacteria and fungi, respectively. Our observations highlight the stability of the indoor airborne bacterial diversity over time, while the corresponding eukaryote community was less stable. Bacterial diversity characterized by pyrosequencing 454 showed high diversity dominated by the Proteobacteria which represented 51.1%, 46.9%, and 38.4% of sequences, for each of the three air samples sequenced. A common bacterial diversity was underlined, corresponding to 58.4% of the sequences. The core species were belonging mostly to the Proteobacteria and Actinobacteria, and to the genus Paracoccus spp., Acinetobacter sp., Pseudomonas sp., Enhydrobacter sp., Sphingomonas sp., Staphylococcus sp., and Streptococcus sp.