The aim of this study was to compare the efficacy of using a dishwasher or different chemical agents, including 0.12% chlorhexidine gluconate, 2% sodium hypochlorite (NaOCl), a mouthrinse containing essential oils and alcohol, and 50% white vinegar, for toothbrush disinfection. Sixty volunteers were divided into five experimental groups and one control group (n = 10). Participants brushed their teeth using toothbrushes with standard bristles, and they disinfected the toothbrushes according to instructed methods. Bacterial contamination of the toothbrushes was compared between the experimental groups and the control group. Data were analyzed by Kruskal–Wallis and Duncan's multiple range tests, with 95% confidence intervals for multiple comparisons. Bacterial contamination of toothbrushes from individuals in the experimental groups differed from those in the control group (p < 0.05). The most effective method for elimination of all tested bacterial species was 50% white vinegar, followed in order by 2% NaOCl, mouthrinse containing essential oils and alcohol, 0.12% chlorhexidine gluconate, dishwasher use, and tap water (control). The results of this study show that the most effective method for disinfecting toothbrushes was submersion in 50% white vinegar, which is cost-effective, easy to access, and appropriate for household use.