Removal of dental plaque and local application of local chemical adjuncts, such as chlorhexidine (CHX), have been used to control and treat peri‐implant disease. However, these methods can damage the surface properties of the implants or promote bacterial resistance. The application of ozone as an adjunctive treatment represents a new approach in the management of peri‐implantitis. Thus, the purpose of this study was to evaluate the antimicrobial effect of ozonized physiological saline solution in different concentrations against oral biofilms developed on titanium surface.

Single and multi‐species biofilms of Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus oralis were formed on titanium specimens for 5 days in anaerobic conditions. Biofilms were treated with ozonized saline solution at different concentrations (25, 50, and 80 μg/NmL), for 30 seconds and 1 minute. CHX (0.12%) and saline solution (0.89% NaCl) were used as positive and negative controls, respectively. Bacterial viability was quantified by colony forming units (CFU mL⁻¹), and biofilm images were acquired by confocal laser scanning microscopy (CLSM). Data were analyzed by parametric test (ANOVA) with Tukey post‐hoc test (P < 0.05).

Ozonized saline solution showed antibiofilm activity at a concentration of 80 μg/NmL for 30 seconds and 1 minute, reducing, mainly, Porphyromonas gingivalis viability, with 2.78 and 1.7 log₁₀ CFU mL⁻¹ of reduction in both single and multi‐species biofilms, respectively, when compared to the control (saline), whereas CHX reduced 1.4 and 1.2 log₁₀ CFU mL⁻¹.

Ozonized saline solution has antibiofilm activity, with better effect when applied for 1 minute at 80 μg/NmL, being a promising candidate therapy for the treatment of peri‐implant diseases.