Objective: The aim of this study was to evaluate the efficacy of a spray containing an antimicrobial solution for toothbrush disinfection. Methods and Materials: Three different solutions were sprayed on toothbrush bristles among 30 adults after they had brushed: (1) basic formulation (base) plus chlorhexidine; (2) base only, and (3) sterile tap water (control). Each solution was tested for 1 week. After that, the toothbrushes were collected and sonicated in Letheen Broth, diluted in 10-fold series, and plated on selective and nonselective media for detection of anaerobes, aerobes, streptococci, and gram-negative bacilli. After incubation, the colonies of those microorganisms were counted. Presence of mutans streptococci on the bristles was also confirmed. Results: Spray 1 produced a significant reduction in the microbial contamination of toothbrushes for all the microorganisms, spray 2 provided some reduction of contaminants, and spray 3 demonstrated the least antimicrobial effect. Conclusion: The antimicrobial spray with chlorhexidine proved to be an effective and practical means for toothbrush disinfection. Keywords: antimicrobial agent, bacterial contamination, biofilm, chlorhexidine, disinfection, oral hygiene aids, spray, toothbrush