This study investigates the microflora of rare manuscripts preserved in the historical monuments of Samarkand city, specifically in the Sherdor Madrasa and the Ulugbek Observatory. The research identified the primary bacterial species responsible for the deterioration of manuscripts based on the density and enzymatic activity of heterotrophic bacteria present in the samples. Microbiological analysis of the damaged manuscripts revealed the presence of bacteria belonging to the genus Bacillus, and fungi of the genera Fusarium, Alternaria, Verticillium, Aspergillus, and Penicillium. All nine isolates exhibiting enzymatic activity were examined at the molecular level. The identified species included Bacillus sp., Bacillus aryabhattai, Bacillus mojavensis, Bacillus simplex, Bacillus halotolerans, Bacillus tequilensis, and Bacillus subtilis, all belonging to the family Bacillaceae