Objective: To assess the antibacterial effect of 6% hydrogen peroxide (H2 O2 ) compared with 1% and 2% sodium hypochlorite, on toothbrushes inoculated with Streptococcus mutans atcc® 25175™. Materials and methods: The study design was an experimental in vitro, cross-sectional prospective and comparative study. Sixty toothbrushes were used, which were divided into four groups of 15 brushes. After conducting a pilot study, the sample number was determined by the means comparison formula and these were selected by simple random sampling. These brushes were inoculated with strains of S. mutans atcc® 25175™. The disinfectants included H2 O2 at 6% and sodium hypochlorite (NaOCl) at 1% and 2%. The Shapiro–Wilk test was used to assess normality and the Mann-Whitney U test was applied for the hypothesis test. Results: The 6% H2 O2 showed an average growth of 2 × 109 cfu/mL, which indicates that its effectiveness is greater compared to the 1% NaClO that showed a growth average of 4 × 109 cfu/mL which is less than the 2% NaClO that presented 0 cfu/mL of S. mutans (p = 0.004). Conclusion: Between 6% H2 O2 and 1% and 2% NaClO, specifically antibacterial detection, 2% NaClO was concluded as being the most effective.