As the largest and best-preserved wooden ancient vessel of the Qing Dynasty in China, the Yangtze River Estuary No.2 shipwreck carries rich marine archaeological and historical-cultural value, making its conservation work of great importance and urgency. In this study, X-ray detection and analysis were employed to clarify the distribution characteristics of iron nails within the wood. By combining high-throughput microbial sequencing, genomics and metabolomics technologies, the structural and functional characteristics of the pathogenic microbial community on the ship hull were analyzed, and the results indicated that the deterioration of the wooden hull might contribute to various mechanisms such as iron oxidation, acidic metabolite production, and cellulase secretion. The results of genomic analysis showed that the bacterial community was dominated by the core dominant genera Acidovorax and Hydrogenophaga, while the fungal community was mainly composed of the saprophytic genera Fusarium and Trichoderma. Functional prediction and metabolite identification revealed that the bacterial community’s core metabolic pathways focus on carbohydrate and amino acid metabolism, with a rich diversity of organic acid compounds in metabolites, additionally iron ion transport and redox-related pathways were enriched in severely corroded samples. The fungal community exhibits a clear dominance of saprophytic types, forming a synergistic degradation effect with pathogenic types. These findings preliminarily elucidate the molecular biological mechanisms of iron component corrosion and microbial synergistic interactions, providing a scientific basis for investigating the degradation mechanisms of the Yangtze River Estuary No.2 shipwreck.