Aim: Serratia marcescens is an important multidrug-resistant human pathogen. The pathogenicity of S. marcescens mainly depends on the quorum sensing (QS) mechanism, which regulates the virulence factors production and biofilm formation. Hence, targeting QS mechanism in S. marcescens will ultimately pave the way to combat its pathogenicity. Thus, the present study is intended to evaluate the efficacy of Vetiveria zizanioides root extract-mediated silver nanoparticles (AgNPs) as a potent anti-QS and antibiofilm agent against S. marcescens. Methods and Results: The AgNPs were synthesized using V. zizanioides aqueous root extract and the physiochemical properties of V. zizanioides-based AgNPs (VzAgNPs) were evaluated using analytical techniques such as ultraviolet-visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering and scanning and transmission electron microscopic techniques. VzAgNPs were found to attenuate the QS-dependent virulence factors, namely prodigiosin, protease, lipase, exopolysaccharide productions and biofilm formation of S. marcescens, without inhibiting its growth. Further, the transcriptomic analysis confirmed the down-regulation of QS-dependent genes, which encode for the production of virulence factors and biofilm formation. Conclusion: The current study confirms VzAgNPs as an ideal anti-QS and antibiofilm agent against S. marcescens. Significance and Impact of the Study: This is the first approach that validates the anti-QS and antibiofilm potential of phytosynthesized VzAgNPs against the nosocomial pathogen, S. marcescens. As VzAgNPs exhibits potent antivirulent activities, it could be used to treat hospital-acquired S. marcescens infections.