The prevalence of silver nanoparticles (AgNPs) in freshwater environments has long been the subject of concern due to their negative impacts on macro-organisms in general. Nevertheless, it is still not well established how freshwater biofilm microbial communities respond to AgNP exposure. This study employed modelled freshwater biofilms to study their response and interaction with AgNPs by applying metagenomic and metabolomic approaches and high-resolution microscopy. Metagenomic 16 S rRNA sequencing analyses revealed that biofilm microbial diversity did not alter following AgNP exposure (p > 0.05). Further genotype investigation highlighted two key taxa, namely Alphaproteobacteria and Betaproteobacteria, as the backbone of the multispecies biofilm when exposed to AgNPs, which also explained increased extracellular polymeric substance (EPS) production. LS-MS/MS-based untargeted metabolomics demonstrated the role of glycerophospholipid metabolism in maintaining biofilm. Scanning electron microscopic analysis revealed distinct biofilm structural phenotypes characterized by smoother biofilm features exposed to AgNPs. Overall, the study presents a unique view of how multispecies biofilms respond to single environmental stress in the form of silver nanoparticle exposure.