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Objectives: The aim of this randomized split‐mouth clinical trial was to evaluate the effects of ozone therapy on clinical and biochemical parameters of moderate to severe generalized periodontitis patients after non‐surgical periodontal therapy. Methods: A total of 36 moderate to severe generalized periodontitis patients were included in the study. The patients were systemically healthy and 18 to 64 years of age. Periodontal parameters, including plaque index (PI), gingival index (GI), probing depth (PD), percentage of bleeding on probing, percentage of pockets deeper than 5 mm and clinical attachment level (CAL), and percentage of ≥3 mm CAL, were evaluated at baseline and 3 months following periodontal therapy. All participants were treated non‐surgically. Topical gaseous ozone was applied into periodontal pockets twice a week for 2 weeks during active periodontal therapy. Gingival crevicular fluid pentraxin‐3 (PTX‐3), interleukin‐1β (IL‐1β), and high sensitivity C‐reactive protein (Hs‐CRP) were evaluated. All statistical data were analyzed using SPSS software. Results: Total of 36 participants completed the study (18 males, 18 females). PI, GI, PD, percentage of bleeding on probing, percentage of pockets deeper than 5 mm and CAL, and percentage of ≥3 mm CAL were improved, and there were no significant differences between the two sides. All inflammatory parameters, PTX‐3, Hs‐CRP, and IL‐1, were reduced at 3‐month follow‐up. Only the decrease in PTX‐3 levels between baseline and 3‐month follow‐up was statistically significant. Conclusions: Ozone therapy did not have any additional effect on periodontal parameters. All cytokines were reduced after periodontal therapy. Only PTX‐3 levels were significantly lower at ozone sites compared to those at the control sites. [ABSTRACT FROM AUTHOR]