The field of green synthesis, namely using plant extracts for the production of metal nanoparticles, is rapidly gaining traction. Therefore, this study investigated the process of producing zinc oxide nanoparticles (ZnO NPs) using a water-based extract derived from the stem bark of Calophyllum teysmannii. Notably, this is the first documented utilization of this particular plant source. The presence of a distinct Ultraviolet-Visible (UV-Vis) absorption peak at 372 nm provided evidence for the creation of ZnO nanoparticles. The X-ray Diffractometer (XRD) and Field Emission Scanning Electron Microscopy (FESEM) investigations indicated that the nanoparticles exhibited sizes ranging from 31.5 to 59.9 nm and had spherical morphologies. Energy Dispersive X-ray Diffractometer (EDX) analysis verified the elemental composition of the ZnO nanoparticles, whereas the Fourier Transform Infrared (FTIR) spectra showed clear peaks, demonstrating their production. The FTIR examination of the C. teysmannii extract revealed peaks at around 3370 cm, indicating the presence of phenolic compounds. These chemicals are likely responsible for the reduction and stabilization of the ZnO NPs. The high-resolution X-ray Photoelectron Spectroscopy (XPS) spectra clearly revealed separate peaks corresponding to Zn 2p and O 1s, providing confirmation of the chemical states and bonding contexts. The Raman Spectroscopy analysis revealed a distinct peak at around 425 cm⁻¹, confirming the presence of the wurtzite structure. The harmful effects of ZnO nanoparticles on HCC2998 (a kind of human colon cancer) and Vero (a type of monkey kidney epithelial) cells were evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT), dichlorodihydrofluorescein diacetate (DCFH-DA), and boron-Dipyrromethene (BODIPY) assays. The cancer cells underwent cell death due to oxidative stress in a dose-dependent manner, as confirmed by microscopic and flow cytometry investigations.