Zinc oxide nanoparticles (ZnO NPs) exhibit selective toxicity to prokaryotic and eukaryotic systems. ZnO NPs (~13 nm) inhibited the growth of Escherichia coli (E. coli) at concentrations ≥3.4 mM and Staphylococcus aureus (S. aureus) at ≥1 mM. Flow cytometry assays confirmed that ZnO NP toxicity was accompanied by a loss of cell viability. However, ZnO NPs had minimal effects on primary human T cell viability, even at concentrations toxic to both gram-negative and gram-positive bacteria. These findings suggest that ZnO NPs selectively target bacterial systems and human T lymphocytes. ZnO NPs, with sizes in the 1–100 nm range, are attractive for biomedical applications due to their potential for selective toxicity. However, many benign materials become toxic at the nanoscale. The study investigated the toxicity of ZnO NPs to E. coli, S. aureus, and human T cells. ZnO NPs caused cell death in both bacterial species, with E. coli showing bacteriocidal toxicity at 5 mM and S. aureus at 1 mM. In contrast, human T cells showed significantly higher tolerance to ZnO NPs, with viability remaining above 77% at 5 mM and 43% at 10 mM. The study also compared the toxicity of ZnO NPs to bulk ZnO powder. While bulk ZnO had no effect on T cell viability, ZnO NPs caused significant decreases in viability at higher concentrations. These results indicate that ZnO NPs are more toxic to bacteria than to human T cells. The selectivity of ZnO NPs in their toxic effects suggests potential applications in biomedical and antibacterial treatments. The findings highlight the importance of understanding NP toxicity to develop safe and effective nanomedicine applications.Zinc oxide nanoparticles (ZnO NPs) exhibit selective toxicity to prokaryotic and eukaryotic systems. ZnO NPs (~13 nm) inhibited the growth of Escherichia coli (E. coli) at concentrations ≥3.4 mM and Staphylococcus aureus (S. aureus) at ≥1 mM. Flow cytometry assays confirmed that ZnO NP toxicity was accompanied by a loss of cell viability. However, ZnO NPs had minimal effects on primary human T cell viability, even at concentrations toxic to both gram-negative and gram-positive bacteria. These findings suggest that ZnO NPs selectively target bacterial systems and human T lymphocytes. ZnO NPs, with sizes in the 1–100 nm range, are attractive for biomedical applications due to their potential for selective toxicity. However, many benign materials become toxic at the nanoscale. The study investigated the toxicity of ZnO NPs to E. coli, S. aureus, and human T cells. ZnO NPs caused cell death in both bacterial species, with E. coli showing bacteriocidal toxicity at 5 mM and S. aureus at 1 mM. In contrast, human T cells showed significantly higher tolerance to ZnO NPs, with viability remaining above 77% at 5 mM and 43% at 10 mM. The study also compared the toxicity of ZnO NPs to bulk ZnO powder. While bulk ZnO had no effect on T cell viability, ZnO NPs caused significant decreases in viability at higher concentrations. These results indicate that ZnO NPs are more toxic to bacteria than to human T cells. The selectivity of ZnO NPs in their toxic effects suggests potential applications in biomedical and antibacterial treatments. The findings highlight the importance of understanding NP toxicity to develop safe and effective nanomedicine applications.