A cheese processing facility seeking to reduce environmental Listeria colonization initiated a regime of ozonation across all production areas as an adjunct to its sanitation regimes. A total of 360 environmental samples from the facility were tested for Listeria over a 12-month period. A total of 15 areas before and 15 areas after ozonation were tested. Listeria isolations were significantly (P < 0.001) reduced from 15.0% in the preozonation samples to 1.67% in the postozonation samples in all areas. No deleterious effects of ozonation were noted on the wall paneling, seals, synthetic floors, or cheese processing equipment. The ozonation regime was readily incorporated by sanitation staff into the existing good manufacturing practice program. The application of ozone may result in a significant reduction in the prevalence of Listeria in food processing facilities.