Palm-leaf manuscripts are precious ancient documents that record religious scriptures, literature, and other content using palm leaves as the writing medium. These manuscripts are primarily preserved and circulated in Xizang and Yunnan, China. Due to long-term preservation and environmental factors, some palm-leaf manuscripts have been contaminated by molds to varying degrees. To understand the extent of mold infestation and identify effective prevention and removal methods, samples were collected from two museums in Xizang and Yunnan for mold observation, strain analysis, and identification. Molds on the surface of palm-leaf manuscripts appeared in black, white, and gray colors and were characterized by different sizes and irregular shapes. In-situ microscopic observation was conducted using an ultra depth-of-field microscope and a scanning electron microscope. Some molds, such as Chaetomium, exhibited typical morphological characteristics. The result of scanning electron microscopy revealed a significant presence of mycelia and spores, invisible to the naked eye, in areas surrounding the visible mold, indicating a strong tendency for mold to spread and thus a need for timely observation and treatment. Sampling, culturing, isolation, and identification were performed on eight samples from the two museums. A total of 14 strains were isolated and were identified—based on microscopic morphology and ITS sequence comparison—as belonging to five genera:Penicillium, Alternaria, Aspergillus, Chaetomium, and Cladosporium. For manuscripts from Xizang, Penicillium was the most common, followed by Alternaria and Chaetomium. For Yunnan samples, Aspergillus was more abundant, followed by Cladosporium and Alternaria. Most strains are common in nature and pose certain risks to organic materials. Mold contamination was attributed to the high humidity of the manuscripts and their storage environments. Differences in microbial types and quantities, along with varying restoration and preservation methods, contributed to the variation in contamination levels between the two regions. In addition, three commonly used natural plant essential oils—citronella oil, clove oil, and eucalyptus oil—were tested for antifungal effects on the five isolated mold types using the disk diffusion method. Results show that citronella oil had the best antifungal effect on all the five mold types, followed by clove oil. Eucalyptus oil exhibited a strong antifungal effect only on Chaetomium globosum but was less effective against the other molds. This study employed multiple analytical methods to characterize, isolate, and identify molds on palm-leaf manuscripts and assessed the antifungal properties of three essential oils. These findings contribute to the understanding of mold-related deterioration and provide a scientific foundation for future treatments and preventive measures.