Development of new nanomaterials that inhibit or kill bacteria is an important and timely research topic. For example, financial losses due to infectious diseases, such as diarrhea, are a major concern in livestock productions around the world. Antimicrobial nanoparticles (NPs) represent a promising alternative to antibiotics and may lower antibiotic use and consequently spread of antibiotic resistance traits among bacteria, including pathogens. Four formulations of zinc nanoparticles (ZnA, ZnB, ZnC, and ZnD) based on phosphates with spherical (ZnA, ZnB) or irregular (ZnC, ZnD) morphology were prepared. The highest in vitro inhibitory effect of our NPs was observed against Staphylococcus aureus (inhibitory concentration values, IC50, ranged from 0.5 to 1.6 mmol/L), followed by Escherichia coli (IC50 0.8–1.5 mmol/L). In contrast, methicillin resistant S. aureus (IC50 1.2–4.7 mmol/L) was least affected and this was similar to inhibitory patterns of commercial ZnO-based NPs and ZnO. After the successful in vitro testing, the in vivo study with rats based on dietary supplementation with zinc NPs was conducted. Four groups of rats were treated by 2,000 mg Zn/kg diet of ZnA, ZnB, ZnC, and ZnD, for comparison two groups were supplemented by 2,000 mg Zn/kg diet of ZnO-N and ZnO, and one group (control) was fed only by basal diet. The significantly higher (P < 0.05) Zn level in liver and kidney of all treated groups was found, nevertheless Zn NPs did not greatly influence antioxidant status of rats. However, the total aerobic and coliform bacterial population in rat feces significantly decreased (P < 0.05) in all zinc groups after 30 d of the treatment. Furthermore, when compared to the ZnO group, ZnA and ZnC nanoparticles reduced coliforms significantly more (P < 0.05). Our results demonstrate that phosphate-based zinc nanoparticles have the potential to act as antibiotic agents.