Background: Toothbrushes may play a significant role in plaque control. Toothbrushes should be correctly stored, disinfected and changed at regular intervals. Objective: The purpose of this study was to evaluate the efficacy of 0.2% chlorhexidine (CHX) gluconate solution and ultra violet (UV) toothbrush‑sanitizer for toothbrush disinfection. Materials and Methods: Fresh tooth brushes were distributed to fifteen study subjects, who were selected randomly and who met the study criteria. All the study participants were asked to brush their teeth with the tooth brush provided. No special instructions were given regarding the brushing techniques. Toothbrushes were collected after 7 days. All tooth brushes were randomly allocated to three groups. Tooth brushes were subjected to microbial analysis and total bacterial count was assessed. Tooth brushes allocated to Group I were soaked in 2% CHX mouthwash for 12 h, Group II were kept in UV‑light toothbrush holder for 7 min, and Group III were soaked in normal saline for 12 h. All the toothbrushes were subjected for microbial analysis and mean bacterial count was determined. Results: There was a statistically significant difference between mean colony‑forming unit count pre‑sanitization and post‑sanitization in all the groups, using 0.2% CHX gluconate, UV rays and normal saline (P < 0.007). However, the mean bacterial count reduced drastically after the treatment with UV rays (P = 0.001). Conclusions: CHX, UV rays and normal saline are effective in a reduction of bacterial count on toothbrushes. UV rays treatment was more effective, when compared to CHX and normal saline.