Aims: To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7. Methods and Results: ZnO NP with sizes of 70 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ and NP‐free solutions were used in antimicrobial tests against E. coli O157:H7. ZnO NP showed increasing inhibitory effects on the growth of E. coli O157:H7 as the concentrations of ZnO NP increased. A complete inhibition of microbial growth was achieved at the concentration level of 12 mmol l⁻¹ or higher. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy were used to characterize the changes of morphology and cellular compositions of bacterial cells treated with ZnO NP and study the mode of action of ZnO NP against E. coli O157:H7. The intensity of lipid and protein bands in the Raman spectra of bacterial cells increased after exposure to ZnO NP, while no significant changes in nucleic acid bands were observed. Conclusions: ZnO NP were found to have antibacterial activity against E. coli O157:H7. The inhibitory effects increase as the concentration of ZnO NP increased. Results indicate that ZnO NP may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells. Significance and Impact of the Study: These results suggest that ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety.