Background: The importance of preserving and maintaining printed materials is crucial for the libraries. Fungi play the main role in destroying wood and paper..Objectives: This research aimed to study and identify threatening fungal agents of library resources in Isfahan University of Medical Sciences..Materials and Methods: This is a descriptive analytical study. 126 samples were collected and examined for the presence of fungi. An open plate method was used to scan airborne fungal contents and triplicate samples were collected at four different locations in the morning, at noon, and in the evening. The fungal culture media were incubated at 25-30 oC until growth appeared and then the fungi colonies were identified by routine mycological laboratory methods..Results: 1265 colonies of fungi belonging to 26 genera were identified in the air and different surfaces of books (references and circulation departments) and also surfaces of shelves in libraries. Cladosporium sp., Penicillium sp., Aspergillus sp. and Alternaria sp. were the most common isolated fungi in libraries of Isfahan University of medical sciences. .Conclusions: We suggest training librarians as one of the most important steps in libraries to preserve library materials because having no knowledge about threatening factors and the way to fight with them are the main reasons of most frequent damages to library resources. Using new methods and technologies of preserving and maintenance of materials should be a priority in library managers’ planning .