Liver and breast cancer are the most traumatic diseases because they affect the major organs of the body. Nanomedicine recently emerged as a better option for the treatment of these deadly diseases. As a result, many nanoparticles have been used to treat cancer cell lines. Of the various nanoparticles, zinc oxide exhibits biocompatibility. Therefore, the aim of the present study was to investigate the activity of zinc oxide nanoparticles (ZnO-NPs) against HepG2 and MCF-7 cells. The NPs (∼13±2 nm) were prepared via a non-protonated chemical route and were well-characterized through standard techniques. The study showed that treatment with NPs is notably effective against the proliferation of HepG2 and MCF-7 cancer cells in a dose-dependent manner. The MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a tetrazole) assays revealed the concentration-dependent cytotoxic effects of NPs in range of 2.5-100 μg/ml. HepG2 and MCF-7 cells were exposed to ZnO-NPs and exhibited a significant reduction in their cell viability (95% and 96%; p<0.05) in response to a very low concentration (25 μg/ml) of the ZnO-NPs; this finding was confirmed with FACS (fluorescence-activated cell sorting) data. The reduction in cell viability in response to NP treatment induces cytotoxicity in the cultured cells. The quantitative RT-PCR (real-time polymerase chain reaction) results demonstrate that the exposure of HepG2 cells to ZnO-NPs results in significant upregulation of the mRNA expression level of Bax, p53, and caspase-3 and the down regulation of the anti-apoptotic gene Bcl-2. The NPs were also tested against five pathogenic bacteria through the disk diffusion method, and their antibacterial activities were compared with that of ZnO salt.