A novel in-package ozonation device was evaluated for its efficacy in inactivating three microorganisms (viz., Listeria innocua, attenuated Salmonella Typhimurium, and Escherichia coli O157:H7) on tomatoes and for its effect on fruit quality. The device produced ozone inside sealed film bags, reaching a concentration of 1,000 ppm within 1 min of activation. The three bacterial cultures were inoculated onto either the smooth surface or the stem scar areas of the tomatoes, which were then sealed in plastic film bags and subjected to in-package ozonation. L. innocua on tomatoes was reduced to nondetectable levels within 40 s of treatment on the tomato surface, with inactivation of ca. 4 log CFU per fruit on the stem scar area. An increase in treatment time did not result in a proportional increase in bacterial reduction. For E. coli O157:H7 and Salmonella, there was little difference (<1 log) in the effectiveness of the system when comparing surface and scar-inoculated bacteria. Both bacteria were typically reduced by 2 to 3 log CFU per fruit after 2- to 3-min treatments. No negative effects on fruit color or texture were observed during a 22-day posttreatment storage study of ozone-treated tomatoes. These results suggest that the three bacteria responded differently to ozonation and that in-package ozonation may provide an alternative to chemical sanitizers commonly used by the industry.