Green synthesis of nanoparticles (NPs) has garnered wide research interest due to inherent properties such as eco-friendliness, compatibility with substrates, and cost-effectiveness. Here, zinc oxide nanoparticles (ZnO-NPs) were successfully synthesized for the first time using Capsicum chinense fruit extract. The optical property of the green and conventionally synthesized ZnO-NPs was characterized by UV-vis spectrophotometer, which exhibited absorption peaks at 302 and 481 nm, respectively, and the morphology of the NPs was analyzed by transmission and scanning electron microscopies (TEM and SEM). The X-ray diffraction (XRD) studies showed that the hexagonal wurtzite phase was obtained, with high crystalline nature, while the electron dispersion X-ray study (EDX) revealed the purity of ZnO-NPs. The cytotoxicity assay of the biosynthesized and conventionally synthesized ZnO-NPs was evaluated using human embryonic kidney (HEK 293) and cervical carcinoma (HeLa) cell lines treated with various concentrations of the ZnO-NPs and they exhibited reasonable activity. Antioxidant activity of the ZnO-NPs was measured using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay and the green ZnO-NPs exhibited higher activity compared to conventional ZnO-NPs. These findings proved that aqueous extracts of C. chinense fruit are effective for the biosynthesis of ZnO-NPs with anticancer and antioxidant potential.