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 usingCassia siamea(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@Cs-NPs to suppress quorum-mediated virulence factors and biofilm formation against clinical MDR isolates (Pseudomonas aeruginosaPAO1 andChromobacterium violaceumMCC-2290) was tested. The ½MIC of ZnO@Cs-NPs reduced violacein production byC. violaceum. Furthermore, ZnO@Cs-NPs sub-MIC significantly inhibited virulence factors such aspyoverdin, pyocyanin, elastase, exoprotease, rhamnolipid, and the swimming motility ofP. aeruginosaPAO1 by 76.9, 49.0, 71.1, 53.3, 89.5, and 60%, respectively. Moreover, ZnO@Cs-NPs also showed wide anti-biofilm efficacy, inhibiting a maximum of 67 and 56% biofilms inP. aeruginosaandC. violaceum, respectively. In addition, ZnO@Cs-NPs suppressed extra polymeric substances (EPS) produced by isolates. Additionally, under confocal microscopy, propidium iodide-stained cells ofP. aeruginosaandC. violaceumshow ZnO@Cs-NP-induced impairment in membrane permeability, revealing strong anti-bacterial efficacy. This research demonstrates that newly synthesized ZnO@Cs-NPs demonstrate a strong efficacy against clinical isolates. In a nutshell, ZnO@Cs-NPs can be used as an alternative therapeutic agent for managing pathogenic infections.