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Background: Toothbrushes that are used regularly can accumulate a high amount of microorganisms, which have the potential to cause infections or reinfections. There is a need for toothbrush disinfection methods which are effective and can be easily implemented. The present study assessed the antimicrobial efficacy of different disinfectants on contaminated toothbrushes in healthy and chronic periodontitis patients. Materials and Methods: Sixty patients were taken who were divided into two Groups A and B: Group A consisted of 30 healthy patients who were further subgrouped as Group A1 (control group), Group A2 (1% sodium hypochlorite), Group A3 (1% vinegar), and Group A4 (ultraviolet [UV] sanitizer) and Group B consisted of 30 patients with mild-to-moderate generalized chronic periodontitis who were further subgrouped as B1, B2, B3, and B4 based on the different disinfection processes. Microbial counts were assessed and compared. A paired sample t-test was used for statistical analysis, and the level of significance was set at P < 0.001. Results: All disinfectants showed a statistically significant reduction in microbial load on contaminated toothbrushes in both the groups (P < 0.001). There was no statistical difference between the efficacies of vinegar and sodium hypochlorite when compared with UV sanitizer in both the groups (P = 0.115 and P = 0.205). Conclusion: The study concluded that UV sanitizer can be used as a preferable toothbrush disinfection method.