Aim: Ozone has been used for biofilm management of carious lesions for a long time. While adjunctive antibacterial strategies are part of periodontal therapy to eliminate or reduce the number of periodontopathogenes, their main focus so far is the application of antiseptic mouthrinses or antibiotics. However, administration of ozone could be beneficial and represent another therapeutic approach. It was the aim of this study to develop an experimental design to test the reduction of Aggregatibacter actinomycetemcomitans (A. a.) und Porphyromonas gingivalis (P. g.) after application of gaseous ozone in vitro. Method: Two strains of A. a. (A. a. 3-2-2 and 10-1-3) and P. g. (P. g. 1711 and P. g. USA) were cultivated under microaerophilic and anaerobic conditions. Strains were diluted to an initial count of 1E+08 bacteria per ml. In group 1, an aliquot of 10 µl bacterial suspension was covered with 50 µl agarose to simulate the existence of saliva in the clinical situation. In groups 2 and 3, an aliquot of 10 µl without agarose was suspended. Gaseous ozone was applied in groups 1 and 2 for 20, 30, 40, 50, 60 und 80 s. Group 3 was the control group that remained untreated. After the treatment, the samples were cultivated again for 7 days, then colony forming units (cfu) were counted. Experiments were repeated 10 times at all time intervals and in each group. Statistics: The significance level was set at 5 %. Data were analysed by one-way ANOVA. Post-hoc the Student-Newman-Keuls-Procedure was used to analyse differences between the three groups. The t-test was applied to find differences between obligate and facultative anaerobes and the two strains of A. a. and P. g. Results: In both test groups, the number of bacteria was reduced significantly by ozone application in comparison to the untreated control group. In group 1, the number of viable bacteria decreased by two log units after an application time of 80 s while this reduction was reached in group 2 already after 60 s. No changes happened in the control group. Clinically meaningful and statistically significant reductions between A. a. und P. g. occurred in group 2 after 80 s only. Differences in between the species were detected. Conclusions: Ozone exhibits an antibacterial effect on A. a. and P. g. in vitro. Covering of bacteria with agarose inhibits this effect.