The increasing number of multidrug resistant bacteria raises a serious public-health concern, which is exacerbated by the lack of new antibiotics. Metal oxide nanoparticles are already applied as an antibacterial additive in various products used in everyday life but their modes of action have remained unclear. Moreover, their potential negative effects to human health are still under evaluation. We explored effects of mixed metal oxide Zn0.15Mg0.85O on Bacillus subtilis, as a model bacterial organism, and on murine macrophages. Zn0.15Mg0.85O killed planktonic bacterial cells and prevented biofilm formation by causing membrane damages, oxidative stress and metal ions release. When exposed to a sub-inhibitory amount of Zn0.15Mg0.85O, B. subtilis up-regulates proteins involved in metal ions export, oxidative stress response and maintain of redox homeostasis. Moreover, expression profiles of proteins associated with information processing, metabolism, cell envelope and cell division were prominently changed. Multimode of action of Zn0.15Mg0.85O suggests that no single strategy may provide bacterial resistance. Macrophages tolerated Zn0.15Mg0.85O to some extend by both the primary phagocytosis of nanoparticles and the secondary phagocytosis of damaged cells. Bacterial co-treatment with ciprofloxacin and non-toxic amount of Zn0.15Mg0.85O increased antibiotic activity towards B. subtilis and E. coli.