Aims This work aimed to evaluate the antimicrobial activity of pure (ZnO) and doped (ZnMgO) zinc oxide (ZnO) nanoparticles on bacterial pathogens and Saccharomyces cerevisiae to confirm their applicability as an alternative to antibiotics and to estimate their biocompatibility. Methods and Results Microbial growth inhibition on agar plates, microbial viability and adaptation tests in broth with ZnO nanoparticles, spore germination, random amplified polymorphic DNA and SDS-PAGE analysis were conducted to evaluate the effects of ZnO nanoparticles on cell morphology, viability, DNA damage and protein production. For this purpose, Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis and S. cerevisiae were studied after the addition of ZnO nanoparticles to the growth media. The contact with ZnO nanoparticles produced changes in morphology, shape, viability, DNA arrangement (DNA fingerprints) and protein content (SDS-PAGE) in treated cells. Conclusions As reported in this study, ZnO nanoparticles have an antimicrobial effect on both prokaryotic and eukaryotic cells. Before using ZnO nanoparticles as antimicrobial agents, it is important to evaluate the target because their effect depends on their composition, size and dose. Significance and Impact of the Study We believe that the results obtained can help to optimize manufactured metal oxide nanoparticles in terms of their composition, size and working concentration. The parameters obtained directly define the applicability and biocompatibility of ZnO nanoparticles and thus are essential for any utilization in food, medicine and industry where pathogen control is crucial.