The manuscripts kept in the manuscript archive of the National Archives and Library of Iran are mostly unique and have special historical and cultural importance. One of the main reasons for the deterioration of manuscripts and books is biological factors, mainly microscopic fungi. The method used in the National Archives and Library of Iran to eliminate biological contamination is disinfection with ethylene oxide. The duration of the disinfecting effects of ethylene oxide gas has always been one of the concerns of this organization and is the aim of this research, during which the disinfecting effect of the mentioned substance has been investigated over a period of 4 years. 107 samples were examined by scraping and culturing in a specific fungal culture medium, and the relationship between their type of contamination with the environmental conditions of storage and the contamination of the manuscripts before disinfection has been discussed. The results showed that although 86.67% of the samples still showed contamination and only 13.43% remained free of contamination, this contamination was different from their initial contamination. Therefore, disinfection with ethylene oxide over a period of 4 years had a good effect because the initial contamination was eliminated and did not grow again after this period of time, and the observed contamination was of the type of environmental contamination that was added to the documents.