The presence of fungal particulate matter in libraries air is a key issue due to its dual impact on the valuable artifacts and on humans. Indoor air fungal composition has been investigated with a volumetric culture-based method only once before in a National Library. The qualitative and quantitative spatiotemporal fluctuations of airborne fungi were evaluated in the National Library of Greece. A portable Burkard sampler was used for the collection of air samples onto Petri dishes with MA2%. Totally, 33 genera of fungi were recovered and identified, with Cladosporium, Penicillium and Aspergillus dominant in indoor air. The genus Aspergillus, in particular, was studied by morphological and molecular analysis, and the prevalent species were assigned to the sections Nidulantes, Nigri and Circumdati. The concentration range of fungal aerosol was 0–2368 CFU/m³ in the first study period and 0–3183 CFU/m³ in the second study period indoors and 51–1997 CFU/m³ outdoors. The mean concentration in indoor air, depending on the site, ranged from 180 to 410 CFU/m³ in the first period and 210–405 CFU/m³ in the second versus 678 CFU/m³ and 516 CFU/m³, respectively, outdoors. The indoor/outdoor concentration ratio (I/O) was 0.5 for total fungi, 0.3 for Cladosporium, 1.1 for Penicillium and 1.4 for Aspergillus. The higher spore concentration and diversity of the genera Penicillium and Aspergillus indoors and the occurrence of less common genera suggest the existence of internal emission sources. The characterization of the fungal community dynamic patterns is an important tool for implementing targeted preventive measures for human safety and conservation of the valuable documentary heritage.