Ozone has a broad antimicrobial spectrum and each microorganism species has inherent sensitivity to the gas. The objective of this study was to evaluate the effect of ozone gas on Escherichia coli O157:H7 inoculated on an organic substrate, and the efficacy of ozonated water in controlling the pathogen. For the first experiment, E. coli O157:H7 (ATCC® 43890™) was inoculated in milk with different compositions and in water, which was ozonated at concentrations of 35 and 45 mg L −1 for 0, 5, 15, and 25 min. In the second experiment, water was ozonated at 45 mg L −1 for 15 min. E. coli O157:H7 was exposed for 5 min to the ozonated water immediately after ozonation, and after storage for 0.5, 1.0, 1.5, 3.0, and 24 h at 8°C. The results showed that the composition of the organic substrate interfered with the action of ozone on E. coli O157:H7. In lactose-free homogenized skim milk, reductions of 1.5 log cycles were obtained for ozonation periods of 25 min at the concentrations tested. Ozonated water was effective in inactivating of E. coli O157:H7 in all treatments. The efficiency of ozone on E. coli O157:H7 is influenced by the composition of the organic substrates, reinforcing the need for adequate removal of organic matter before sanitization. Furthermore, refrigerated ozonated water stored for up to 24 h is effective in the control of E. coli O157:H7.