Multidrug-resistant Pseudomonas aeruginosa poses a significant threat in healthcare settings, particularly in hospital-acquired infections, due to its intrinsic resistance mechanisms and adaptability. This multidrug-resistant bacterium forms biofilms and regulates diverse virulence factors through quorum sensing. The current study involved the acquisition of metal-tolerant bacterial isolates from a metal-contaminated location, which were subsequently assessed for their capacity to produce multifunctional nanocomposites. The biotransformation of the nanocomposites was visually validated by a rapid color change, mediated by the Bacillus cereus SASAK strain, which was confirmed through 16S rRNA sequencing and subsequently deposited in GenBank (MH885570). The biogenic nanocomposite underwent analysis using UV–Vis–NIR spectroscopy, FTIR, XRD, zeta potential analysis, HRTEM, FESEM, and EDX. The minimum inhibitory concentrations (MICs) of 2% Ag-ZnO nanocomposites were found to be 200 µg/mL, and sub-MICs of 2% Ag-ZnO were 100 µg/mL. The sub-MIC of 2% Ag-ZnO inhibited quorum-sensing-associated virulence factors and biofilm formation. Specifically, there was a marked decrease in violacein production (96.25%), swarming motility, and pyocyanin levels (1.87 µg/mL). Furthermore, biofilm formation and extracellular polymeric substance production were reduced by 81.1% and 83.9%, respectively. Extracellular protease activity also decreased, as evidenced by a reduction in zone size from 2.3 cm to 1.8 cm. Overall, these findings highlight the potential of silver-doped zinc oxide nanocomposites as effective anti-virulence agents targeting quorum-sensing pathways in multidrug-resistant P. aeruginosa.