Objective: To qualitatively and quantitatively assess the microbial contamination of tooth brushes preserved in different sanitary settings before and after disinfection with 0.2% chlorhexidine. Materials and Method: The study was carried out in two phases among thirty participants visiting a dental hospital. These participants were assigned to one of the three groups based on the practice of preserving the tooth brush. Group 1: Participant's who preserved their brush outside the bathroom. Group 2: Participant's who preserved their brush within the bathroom without attached toilets. Group 3: Participants who preserved their brush within the bathroom with attached toilets. Participants were given oral hygiene kits containing a brush and paste in the first phase. The brush samples were collected on day thirty for qualitative and quantitative estimation of microbial contamination. In the second phase, participants were requested to rinse their brushes in 0.2% chlorhexidine after brushing and before placing it back. The mean CFU of different bacteria was compared using independent sample t-test and paired sample t-test. Results: The tooth brushes stored outside the bathrooms demonstrated the presence of Candida, Streptococci, Klebsiella, Staphylococcus aureus and Lactobacillus. Pseudomonas, Candida, Streptococci, Staphylococcus aureus and Lactobacillus was demonstrable in the tooth brush samples of group 2. Pseudomonas, Candida, Streptococci, Klebsiella, Staphylococcus aureus, Lactobacillus, Proteus and E.coli were all demonstrable in group 3. However, the disinfection with 0.2% chlorhexidine reduced the microbial load substantially. Conclusion: Rinsing the tooth brush with 0.2% chlorhexidine substantially reduced the microbial contamination in all the three settings.