In recent years, bacterial pathogens have developed resistance to antimicrobial agents that have created a global threat to human health and environment. As a novel approach to combating antimicrobial resistance (AMR), targeting bacteria’s virulent traits that can be explained by quorum sensing (QS) is considered to be one of the most promising approaches. In the present study, biologically synthesized silver nanoparticles derived from Lactobacillus rhamnosus (AgNPs-LR) were tested against three Gram-negative bacteria to determine whether they inhibited the formation of biofilms and triggered the virulence factors controlled by QS. In C. violaceum and S. marcescens, a remarkable inhibition (>70%) of QS-mediated violacein and prodigiosin production was recorded, respectively. A dose-dependent decrease in virulence factors of P. aeruginosa (pyocyanin, pyoverdine, LasA protease, LasB elastase and rhamnolipid production) was also observed with AgNPs-LR. The biofilm development was reduced by 72.56%, 61.70%, and 64.66% at highest sub-MIC for C. violaceum, S. marcescens and P. aeruginosa, respectively. Observations on glass surfaces have shown remarkable reductions in biofilm formation, with less aggregation of bacteria and a reduced amount of extra polymeric materials being formed from the bacteria. Moreover, swimming motility and exopolysaccharides (EPS) was also found to reduce in the presence of AgNPs-LR. Therefore, these results clearly demonstrate that AgNPs-LR is highly effective in inhibiting the development of biofilms and the QS-mediated virulent traits of Gram-negative bacteria. In the future, AgNPs-LR may be used as an alternative to conventional antibiotics for the treatment of bacterial infections after careful evaluation in animal models, especially for the development of topical antimicrobial agents.