Archaeological silk undergoes destructive and irreversible changes during the natural process of decay. However, in-depth studies on the influence of this biological factor are still lacking. Here, a combination of proteomics and metabolomics is proposed for the first time to explore the interaction between bacteria and historical silk during biodegradation, which provides information on changes at the molecular level of proteins and bacterial metabolites. Morphological observation revealed biofilms produced by Stenotrophomonas maltophilia and Pseudomonas alcaligenes when cultured in the stationary phase and confirmed severe deterioration of silk. Proteomics showed that S. maltophilia had an unbiased effect on silk fibroin, indicating its ability to disrupt both heavy and light chains, as well as other proteins, while P. alcaligenes showed an affinity for more disordered proteins. Analysis of bacterial metabolites showed that overall activity reduction and significant accumulation of fatty acid and phenol metabolites occurred after silk addition, suggesting that the presence of silk may inhibit the activity of an individual strain. This study provides a new insight into the microbial degradation mechanism of archaeological silk.