This study was aimed to fill the critical gap of knowledge regarding the interaction between green zinc oxide nanoparticles (ZnONPs) and bacterial interface. Wurtzite phase ZnONPs with a band gap energy of 3.28 eV were produced by exploiting a simple and green biosynthesis method using an inexpensive precursor of A. indica leaf extract and zinc nitrate. ZnONPs were characterized using UV-Vis spectroscopy, XRD, FTIR, SEM, EDX, DLS, TEM, and zeta-potential analysis. The primary size obtained was 26.3 nm (XRD) and 33.5 ± 6.5 nm (TEM), whereas, the secondary size was found to be 287 ± 5.2 nm with -32.8 ± 1.8 mV ζ-potential denoting the physical colloid chemistry of ZnONPs. Crystallinity and the spherical morphology of ZnONPs were also evident with some sort of particle agglomeration. ZnONPs retained plant functional groups endorsing their hydrophilic character. The antibacterial and antibiofilm activity of ZnONPs was significant (p ≤ 0.05) and the MIC/MBC against most frequent clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus ranged from 0.5 to 1.0 (MIC)/1.0 to 1.5 mg ml-1 (MBC). The dissolution of ZnONPs to Zn2+ ions in a nutrient medium increased as a result of interaction with the bacterial surface and metabolites. Substantial surface binding of ZnONPs followed by intracellular uptake disrupted the cell morphology and caused obvious injury to the cell membrane. Interrupted bacterial growth kinetics, loss of cell respiration, enhanced production of intracellular ROS, and the leakage of the cytoplasmic content unequivocally suggested a strong interaction of ZnONPs with the exterior cell surface and intracellular components, eventually leading to cell death and destruction of biofilms. Overall, the results elucidated eco-friendly production of ZnONPs expressing a prominent interfacial correlation with bacteria and hence, prospecting the use of green ZnONPs as effective nanoantibiotics.