Objective: To evaluate, in vitro, the antimicrobial effect of ozone gas on strains of reference mutans streptococci and strains isolated from children at a high risk of developing caries. Method and Materials: A series of dilutions was obtained from a total of 41 strains, and the effect of ozone was determined for applications of 10, 20, and 40 seconds, as well as a control, in terms of the reduction of viable bacteria. Results: Ozone application for 10 and 20 seconds produced a significant reduction in the number of bacteria, inversely proportional to the bacterial concentration. When exposure lasted 40 seconds, no viable bacteria were obtained. Conclusions: Both time of application and initial bacterial concentration bear an influence on the antimicrobial effect of ozone on mutans streptococci. Keywords: antimicrobial agents, caries, caries therapy, in vitro study, mutans streptococci, ozone