The study investigates the selective toxicity of zinc oxide (ZnO) nanoparticles (NPs) to both prokaryotic and eukaryotic systems, specifically *Escherichia coli* (E. coli) and *Staphylococcus aureus* (S. aureus), as well as primary human T lymphocytes. ZnO NPs, approximately 13 nm in size, were found to completely inhibit the growth of E. coli at concentrations ≥ 3.4 mM and S. aureus at concentrations ≥ 1 mM. Flow cytometry-based assays confirmed that these inhibitory effects were accompanied by a loss of cell viability. In contrast, ZnO NPs had minimal effects on primary human T cell viability at concentrations that were toxic to both bacteria. The study demonstrates the selective toxicity of ZnO NPs to different biological systems and suggests that this property could be exploited for biomedical and antibacterial applications. The findings highlight the importance of understanding the mode and range of NP toxicity to develop targeted and safe applications.The study investigates the selective toxicity of zinc oxide (ZnO) nanoparticles (NPs) to both prokaryotic and eukaryotic systems, specifically *Escherichia coli* (E. coli) and *Staphylococcus aureus* (S. aureus), as well as primary human T lymphocytes. ZnO NPs, approximately 13 nm in size, were found to completely inhibit the growth of E. coli at concentrations ≥ 3.4 mM and S. aureus at concentrations ≥ 1 mM. Flow cytometry-based assays confirmed that these inhibitory effects were accompanied by a loss of cell viability. In contrast, ZnO NPs had minimal effects on primary human T cell viability at concentrations that were toxic to both bacteria. The study demonstrates the selective toxicity of ZnO NPs to different biological systems and suggests that this property could be exploited for biomedical and antibacterial applications. The findings highlight the importance of understanding the mode and range of NP toxicity to develop targeted and safe applications.