Introduction <p>The purpose of the study is to evaluate the effect of medical ozone therapy on fracture healing in rats.</p> Materials and methods <p>20 male Wistar-Albino rats were randomly divided into two groups as Control Group (<i>n</i> = 10) and Ozone Group (<i>n</i> = 10). The fracture model was created by bilateral femur transverse osteotomy and fixation with an intramedullary Kirschner wire. No medical treatment was applied to the control group, whereas Ozone gas at a dose of 1&#xa0;cc/kg at a concentration of 20&#xa0;µg/ml was administered intraperitoneally to the ozone group for 8 weeks. All groups were sacrificed at the end of the 8th week. Radiological examination was performed by direct radiography of all femurs. The left femurs of both groups were examined histopathologically (Hematoxylin-Eosin), immunohistochemically (BMP-7, Osteocalcin, Osteopontin, TRAP) and histochemically (Masson Trichrome). Biomechanical (3-point bending test) analysis was performed on the right femurs. The liver and kidneys were also examined histopathologically.</p> Results <p>Radiographic (<i>p</i> = 0.008) and histopathological (<i>p</i> = 0.001) examinations revealed that fracture healing scores of the Ozone Group were significantly inferior compared to the Control Group. In the immunohistochemical examination, the positivity scores of BMP-7 (<i>p</i> = 0.009), Osteocalcin (<i>p</i> = 0.001) and Osteopontin (<i>p</i> = 0.023) were statistically significantly lower in the Ozone group compared to the control group, while the TRAP (<i>p</i> = 0.016) positivity score was significantly higher. In histochemical examination, Masson Trichrome positivity was found to be significantly lower in the Ozone group compared to the control group (<i>p</i> &lt; 0.001). Biomechanical analysis revealed that fracture healing was lower in the Ozone group compared to the Control group in parameters Yield Force (<i>p</i> = 0.012), Yield at Elongation (<i>p</i> = 0.030), Maximum Force (<i>p</i> = 0.009), Maximum Elongation (<i>p</i> = 0.023), Maximum Stress (<i>p</i> = 0.045). As a result of the examination of possible side effects on liver (<i>p</i> = 1.000) and kidney (<i>p</i> = 0.181), no statistically significant difference was found between the groups.</p> Conclusion <p>Medical ozone therapy demonstrated a detrimental effect on fracture union, as evidenced by inferior radiological, histopathological, immunohistochemical, histochemical, and biomechanical outcomes. These findings indicate that systemic ozone treatment may adversely influence bone healing processes.</p>