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Fungi are the most important agents of biodeterioration in museums, libraries, and repositories. The objectives of the paper were to evaluate the microbial diversity in biodeteriorated manuscripts located in a repository of the central library of Astan Quds Razavi (AQR) and to estimate the fungal occurrence of the repository air. The sterile cotton swabs and nitrocellulose membranes were used for sampling the manuscripts, while the sedimentation method was used for the microbial sampling of air. To evaluate the biodeteriorative impacts of fungi, fungal spore’s suspension inoculated on paper strips. Fourteen and six fungal isolates were collected from the three different deteriorated substrates and the repository air samples, respectively. Among the fungi isolates, Aspergillus sp. was isolated in high frequency (36%), followed by Penicillium sp. (21/5%) and Altelnania sp. (14%). Fungi species including P. chrysogenum, Cladosporium cladosporioides, Talaromyces diversus, and Aspergillus sp. were isolated from B1 sample as a parchment. The most fungal isolates (53%) in the air repository including Purpureocillium lilacinum, Talaromyces diversus, Cladosporium sp., and Aspergillus sp were achieved from MEA medium. The low number of isolated fungi from repository air can be attributed to the efficiently controlled environment factors of AQR repository. The combination of finding provides some support for the conceptual premise that it could be a direct relationship between the isolated microorganisms from air and those isolated from the manuscripts. The presence of color spots on paper strips can approve the biodeterioration of paper via fungal activities.