Palm leaf manuscripts, as a critical component of cultural heritage, hold immense historical and cultural significance, particularly in South Asia and Southeast Asia. Despite their historical importance, these manuscripts are highly susceptible to environmental factors such as relative humidity (RH), which can significantly influence their physical, mechanical, and chemical properties over time. This study aims to investigate the effects of varying RH levels on the aging behavior of palm leaf manuscripts to identify optimal conditions for their long-term preservation. In this study, palm leaf samples were prepared using traditional methods involving leaves of Corypha umbraculifera L., which were boiled, dried, flattened, and refined. Accelerated aging experiments were conducted at five controlled RH levels (10%, 30%, 50%, 70% and 90%) over 100 days at 25℃. A combination of advanced analytical techniques, including dynamic vapor sorption (DVS), thermomechanical analysis (TMA), Fourier transform infrared spectrometry (FTIR), scanning electron microscopy (SEM), and gloss measurement, was employed to assess the morphological, chemical, and mechanical changes of the manuscripts under these conditions. The findings demonstrate that both excessively dry and highly humid conditions adversely affect the preservation of palm leaf manuscripts. Under low RH conditions (10% and 30%), the samples exhibited significant surface cracking and shrinkage, which severely compromised their mechanical properties, such as hardness and flexural strength. The cracking was particularly severe at 10% RH, leading to irreversible structural damage. Conversely, high RH conditions (70% and 90%) promoted mold growth on the samples, causing substantial changes in appearance, including discoloration and loss of gloss. Mold growth also resulted in increased hygroscopicity and accelerated chemical degradation of cellulose and hemicellulose, as evidenced by diminished characteristic peaks in the FTIR spectra. These chemical alterations correlated with a marked decline in mechanical properties of the manuscripts, including a substantial reduction in flexural strength and modulus. In contrast, samples aged at 50% RH exhibited minimal changes across all parameters. The manuscripts retained their original morphological, chemical, and mechanical characteristics, with no significant alteration in surface appearance, color, gloss, or structural integrity. The mechanical properties, including flexural strength and modulus, showed negligible deterioration, highlighting the stability of the material under these conditions. These results suggest that 50% RH is an optimal condition for the preservation of palm leaf manuscripts, as it effectively balances the risks of desiccation and mold-induced degradation. The study also elucidates the underlying mechanisms of manuscript degradation under varying RH conditions. Low RH environments induce moisture loss, which leads to internal stresses and subsequent surface cracking. These physical damages are accompanied by a loss of flexibility and mechanical strength, rendering the manuscripts brittle and vulnerable to further deterioration. In high RH environments, microbial activity is intensified, with mold producing organic acids and enzymes that accelerate the hydrolytic degradation of cellulose and hemicellulose. This dual process of physical and biochemical degradation severely compromises the manuscripts’ preservation. Furthermore, the research highlights the significance of maintaining stable environmental conditions for preventive conservation. The integration of advanced analytical techniques, such as DVS for hygroscopic behavior analysis and TMA for mechanical performance evaluation, provides a robust methodological framework for assessing the impact of environmental factors on organic cultural heritage. Unlike previous studies primarily focusing on visual and morphological changes, this research delves into the chemical mechanisms of degradation, offering a comprehensive understanding of how relative humidity influences the aging of palm leaf manuscripts. The findings of this study have broader implications for the conservation of organic cultural heritage materials. The identification of 50% RH as the optimal preservation condition offers practical guidance for the storage and display of palm leaf manuscripts in museums, libraries, and archives. By emphasizing the interplay between environmental factors and material properties, this research provides critical data for the formulation of evidence-based conservation strategies. In conclusion, the study underscores the necessity of precise environmental control for the preservation of palm leaf manuscripts. Both excessively low and high RH levels pose significant risks to their longevity, highlighting the importance of maintaining a stable and moderate RH environment. While 50% RH has been identified as the optimal condition, future research should explore the combined effects of temperature, pollutants, and other environmental factors to develop a more comprehensive understanding of the long-term preservation requirements for palm leaf manuscripts.