This study presents an inexpensive, eco-friendly, and simple green synthesis of ZnO nanoparticles using Origanum vulgare extract. These nanoparticles are non-hazardous, environmentally friendly, and cheaper than other methods of biosynthesis. Ongoing research determines the role of phytochemicals in the fabrication and biosynthesis of ZnO NPs and their role in antibacterial activity and biomedical applications. Characterizations by fourier transform infrared spectroscopy (FTIR), diffuse reflectance UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) determine the successful biosynthesis of ZnO NPs. Meanwhile, TEM and X-ray diffraction studies approximated the spherical morphology and crystalline nature of biosynthesized ZnO NPs of nano size in the range of 20–30 nm. The global increase in drug resistance necessitates the search for new drugs with different mechanisms of action. Quorum sensing (QS), a cell-to-cell communication, has gained attention as an emerging drug target. It controls numerous biochemical processes in bacteria, which are essential for their survival and pathogenicity. The potential of nanomedicines has also been tested to synthesize new antibiotics to tackle drug resistance. ZnO NPs were explored for their antibacterial, antiquorum sensing, and antibiofilm activities with a bioreporter strain of Chromobacterium violaceum. Susceptibility testing results indicated the potential antibacterial activity of ZnO NPs with a minimum inhibitory concentration (MIC) of 4 µg/mL and minimum bactericidal concentration (MBC) of 16 µg/mL. Antiquorum-sensing assays revealed that these nanoparticles inhibit quorum sensing with minimum antiquorum sensing activity (MQSIC) of 1 µg/mL, without causing any bacterial growth inhibition. In addition, ZnO NPs inhibit biofilm formation at inhibitory and higher concentrations. RT-qPCR results supported the downregulation of the quorum sensing genes when C. violaceum was treated with ZnO NPs. The outcomes of this study are promising with regard to the biofilm and quorum sensing, emphasizing the potential applications of ZnO NPs against bacterial communication and biofilm formation.