Indoor Air Quality (IAQ) in libraries is influenced by the presence of specific factors which can impact on both paper storage as well as people health. Microclimatic conditions induce and support a biodiversity pattern involving environmental and anthropic microorganisms. We used a multidisciplinary monitoring model to characterize microflora biodiversity by Next Generation Sequencing (NGS). Biodiversity indexes were adapted to evaluate anthropic vs environmental pollution by combining Shannon mean index (H), species representativeness (EH), human/environmental pollution ratio (SA) to better characterize the NGS output and acquire synthetic information on Indoor Air Microbial Biodiversity (IAMB). Results indicate a frequently low microbial load (IGCM/m3 < 1000) characterized by different species (n = 102), including several cellulose metabolizing bacteria. Workers and visitors appeared a relevant source of microbial contamination. Air biodiversity assayed by NGS seems a promising marker for studying IAQ.