This study reports the green synthesis, characterization, and cytotoxic evaluation of zinc oxide (ZnO) nanoparticles. ZnO nanoparticles have been synthesized using 3 mL of waste peel extracted from Allium cepa (onion) and designated as O3. Comprehensive characterization has been performed using XRD, UV–Visible, FTIR, FESEM, HRTEM, DLS, and fluorescence spectroscopy. The UV–Visible spectrum exhibited an absorption peak at 345 nm, confirming ZnO nanoparticle formation. XRD analysis shows a wurtzite hexagonal phase, while FESEM images show rod-shaped morphology with an average length of ∼400 nm and a width of ∼140 nm. FTIR spectra indicated the presence of functional groups in coating of the nanoparticles surface. The efficacy of these nanoparticles for anticancer activity has been evaluated using MTT, crystal violet assay, AO/PI dual staining, flow cytometry, intracellular ROS estimation, and p53 immunocytochemistry. The MTT assay shows dose- and time-dependent cytotoxicity with an IC50 value of 192.63 μg/mL. Crystal violet staining reveals reduced cell density and loss of adherence in treated group. Intracellular ROS estimation using DHE staining shows significant superoxide generation in nanoparticles-treated cells. Flow cytometry demonstrated prominent G1 phase arrest in treated A549 cells, which correlates with increased nuclear p53 expression observed by immunocytochemistry, supporting activation of the p53-dependent checkpoint pathway known to regulate G1 arrest and apoptosis under oxidative stress conditions. Together, these results provide mechanistic support for the apoptosis-inducing effect of the biosynthesized ZnO nanoparticles in A549 lung cancer cells.