Microorganisms remaining beneath restorations can cause secondary caries and pulp damage. Because of this, antimicrobial treatment could be useful. The aim of this study was to evaluate the antibacterial effect of the HealOzone device on Streptococcus mutans and to compare it with the already proven activity of two dentin-bonding systems. Thirty-five human molars were divided into 5 groups. Cavities were then cut into the teeth (n = 28 cavities per group). After sterilization, the teeth were left in broth cultures of 106 colony-forming units (CFU) ml−1 of S. mutans at 36°C for 48 h. The appropriate treatment followed (group A, control; group B, Clearfil SE Bond; group C, Clearfil Protect Bond; group D, 40 s of treatment with ozone; and group E, 80 s of treatment with ozone), and the cavities were then filled with composite resin. After 72 h, the restorations were removed, dentin chips were collected with an excavator, and the total number of microorganisms was determined. All treatments significantly reduced the number of S. mutans present compared with the control group. The antimicrobial effect of both bonding systems and treatment with 80 s of ozone was significantly higher than the 40 s ozone treatment. In conclusion, HealOzone and the bonding systems show striking antimicrobial effects against S. mutans.