Topical application of ozone has been used in management of gingivitis and periodontitis with no reliable data obtained regarding its direct effect on the periodontal tissues in diabetic rats. Blood glucose level measurements were done before and after ozone application. Cementum thickness measurements showed no significant difference in the diabetic groups before and after ozone application. The number of bone cells was also measured showing a significant increase in number as compared with the controls in some of the groups, followed by decrease in number of bone cells with prolonged ozone application. The same results were obtained after counting the number of fibroblasts. The histopathological examination of the samples revealed presence of some structural alterations with prolonged use of ozone. DNA content was measured using the image analysis system showing different degrees of abnormalities denoting the presence of pathological changes.