Objective. This study aimed to evaluate the effectiveness of alternative methods for toothbrush disinfection. Methods. Two-hundred eighty toothbrushes were included in the study. The toothbrushes were divided into 7 groups and were contaminated by standardized suspensions of Lactobacillus rhamnosus (L. rhamnosus), Streptococcus mutans (S. mutans), Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli). The following disinfectants were tested: 1% sodium hypochlorite (NaOCl), 100% and 50% white vinegar, microwave (MW) oven, ultraviolet (UV) sanitizer, and mouth rinse-containing propolis (MCP). Data were analyzed with Kruskal Wallis and Dunn's tests. Results. Statistically significant differences were found between different methods and control group for all tested bacteria. There were statistically significant differences between all test groups for all microorganisms. MW was the most effective for L. rhamnosus and 100% white vinegar was the most effective method for S. mutans and S. aureus. NaOCl was the most effective for E. coli. Conclusion. This study showed that 100% white vinegar was considered to be effective for tested microorganisms. Similarly, 1% NaOCl is cost-effective, easily accessible, and comparatively effective for toothbrush disinfection. Because these agents are nontoxic, cost-effective and easily accessible, they may be appropriate for household use.