The present study fabricates biogenic zinc oxide nanoparticles (ZnO NPs) with the aqueous leaf extract of Annona muricata (Am) plant collected from semi-evergreen forests of Odisha, India. The synthesized Am-ZnO NPs were physicochemically characterized. The ultraviolet/visible spectrum showed the maximum optical absorbance of Am-ZnO NPs at 355 nm. High-resolution transmission electron microscopy analysis presented the nearly spherical shape of Am-ZnO NPs with an average particle size of 80 nm. The net surface charge and hydrodynamic size of Am-ZnO NPs were measured to be ∼−2.59 mV and ∼417 nm, respectively. Am-ZnO NPs were found to be biocompatible and hemocompatible nature. Furthermore, Am-ZnO NPs displayed strong anticancer effects on both 2D and 3D tumor models. We observed a dose-dependent toxicity on both A549 and MOLT4 cells and observed a size reduction in the A549 tumor spheroids. Subsequently, we observed a depolarization in mitochondrial membrane potential of Am-ZnO NP–treated cancer cells leading to the apoptosis induction in cancer cells.