Understanding the impact of mildew on lignin and hemicelluloses within plant cell walls is crucial for addressing biomass degradation and antimicrobial studies. In this work, Two-dimensional nuclear magnetic resonance (2D NMR) and nanoindentation techniques were used to characterize the effects of three mildew species-Aspergillus niger (AN), Trichoderma viride (TV), and Penicillium citrinum (PC)-on lignin and hemicelluloses within bamboo cell walls. 2D NMR detected bond changes, while nanoindentation measured mechanical decline. Results show that mildew infection causes surface discoloration and weakens bamboo's mechanical integrity. Among the three species, AN had the greatest impact, followed by TV and PC. At the macromolecular level, mildew infection reduces the molecular weight of hemicelluloses. Specifically, all three mildew species (AN, PC, and TV) displayed targeted degradation of arabinose side chains in hemicelluloses. Mildew infection also altered the lignin composition in bamboo, with AN and PC infections leading to an elevated syringyl-to-guaiacyl (S/G) ratio, indicating reduced lignin condensation. Notably, TV demonstrated a remarkable ability to selectively cleave resistant carbon‑carbon (β-β) bonds in lignin, which are typically harder to degrade than ester and ether bonds. In summary, this study reveals how mildew strains selectively degrade bamboo lignin and hemicelluloses, offering insights into biomass use and plant-microbe interactions.