Objective: To evaluate in the vitro effectiveness of three chemical agents for toothbrush disinfection. Material and Methods: Sixteen new toothbrushes were evaluated, previously sterilized and classified in five experimental groups (n=3) and one item as control. Three chemical agents were assessed: 0.12% Chlorhexidine gluconate (CHX), essential oil mouth rinse (Listerine) and 3.5% Sodium hypochlorite (NaOCl). The five selected strains were inoculated on toothbrushes and incubated for a 24 hours period and 37°C temperature in aerobic conditions. The incubated toothbrushes were immersed for a 15 min period into selected chemical agents and after drying in a controlled air stream, again re-cultured into enriched broth. A comparison was made between the initial and final microorganisms density recovered after chemical disinfection based on Mc Farland scale. The data obtained was compared by descriptive analysis and ANOVA methodology. Results: 3.5% NaOCl was the most effective chemical agent for toothbrush disinfection followed by CHX; Listerine was not effective to eliminate the inoculated bacteria in toothbrushes. Conclusion: 3.5% NaOCl and 0.12% CHX are the most effective chemical agents for toothbrush disinfection and Listerine was only effective against C. albicans.