Nanoparticles (NPs) have unique properties compared to their bulk counterparts, and they have potentials for various applications in many fields of life science. Green-synthesized NPs have garnered considerable interest due to their inherent features such as rapidity, eco-friendliness and cost-effectiveness. Zinc oxide nanoparticles (ZnO NPs) were synthesized using an aqueous extract of Kalanchoe blossfeldiana as a reducing agent. The resulting nanoparticles were characterized via X-ray diffraction (XRD), dynamic light scattering (DLS), UV-Vis spectroscopy, photoluminescence (PL), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS). The antimicrobial potential of the synthesized ZnO NPs against bacterial and fungal strains was examined by the disk diffusion method, and they showed a promising antibacterial and antifungal potential. The catalytic activity of the synthesized ZnO NPs in reducing methylene blue (MB) and eosin was studied via UV-Vis spectroscopy. The decolorization percentages of the MB and Eosin Y dyes were 84% and 94%, respectively, which indicate an efficient degradation of the ZnO NPs. In addition, the cytotoxic activity of the ZnO NPs on the HeLa cell line was evaluated via in vitro assay. The MTT assay results demonstrate a potent cytotoxic effect of the ZnO NPs against the HeLa cancer cell line.