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The usefulness of nanoparticles (NPs) in biological applications, such as nanomedicine, is becoming more widely acknowledged. Zinc oxide nanoparticles (ZnO-NPs) are a type of metal oxide nanoparticle with an extensive use in biomedicine. Here, ZnO-NPs were synthesized using <i>Cassia siamea</i> (L.) leaf extract and characterized using state-of-the-art techniques; UV–vis spectroscopy, XRD, FTIR, and SEM. At sub-minimum inhibitory concentration (MIC) levels, the ability of ZnO@<i>Cs</i>-NPs to suppress quorum-mediated virulence factors and biofilm formation against clinical MDR isolates (<i>Pseudomonas aeruginosa</i> PAO1 and <i>Chromobacterium violaceum</i> MCC-2290) was tested. The ½MIC of ZnO@<i>Cs</i>-NPs reduced violacein production by <i>C. violaceum</i>. Furthermore, ZnO@<i>Cs</i>-NPs sub-MIC significantly inhibited virulence factors such aspyoverdin, pyocyanin, elastase, exoprotease, rhamnolipid, and the swimming motility of <i>P. aeruginosa</i> PAO1 by 76.9, 49.0, 71.1, 53.3, 89.5, and 60%, respectively. Moreover, ZnO@<i>Cs</i>-NPs also showed wide anti-biofilm efficacy, inhibiting a maximum of 67 and 56% biofilms in <i>P. aeruginosa</i> and <i>C. violaceum</i>, respectively. In addition, ZnO@<i>Cs</i>-NPs suppressed extra polymeric substances (EPS) produced by isolates. Additionally, under confocal microscopy, propidium iodide-stained cells of <i>P. aeruginosa</i> and <i>C. violaceum</i> show ZnO@<i>Cs</i>-NP-induced impairment in membrane permeability, revealing strong anti-bacterial efficacy. This research demonstrates that newly synthesized ZnO@<i>Cs</i>-NPs demonstrate a strong efficacy against clinical isolates. In a nutshell, ZnO@<i>Cs</i>-NPs can be used as an alternative therapeutic agent for managing pathogenic infections.