The study of the mildewing of textile relics plays an important role in their conservation, restoration and storage. In order to identify the microorganisms causing mildewing of textile relics in the collection and investigate the fungal disease characteristics of cotton textile relics, the potato dextrose agar (PDA) medium was used to isolate and purify the microorganisms extracted from the mildewed parts of cotton textile relics. The purified molds were analyzed by 18S rDNA-internal transcribed spacer (ITS) sequence. The National Center for Biotechnology Information (NCBI) sequence database was used to compare sequence homology and construct a developmental tree. Finally, a total of six strains of mold were isolated, purified and identified by sequencing as Penicillium, Alternaria, Aspergillus, and Fusarium. In this study, morphological methods combined with molecular biological measures were used to isolate and identify the microorganisms causing mildewing of cotton textile relics, making the results more rapid and accurate. In combination with the current conservation status of textile relics in museums, the conclusion of comprehensive conservation measures for cotton textile relics was put forward, which could provide a theoretical basis for the mildewing prevention work of such cultural relics and the development of mildewing-resistant materials.