The aim of this study was to evaluate the knowledge and behavior of dentists regarding toothbrush disinfection. This study included 147 dentists (88 women and 59 men) who were actively employed at a dental school in Ankara, Turkey. Participants were asked to fill out a standard questionnaire, which contained questions regarding their demographics, brushing habits, toothbrush storage and disinfection habits, toothpaste use, knowledge about toothbrush disinfection, and whether they advised their patients about toothbrush storage. Descriptive statistics were calculated, and statistical analyses were performed with t-tests, chi-squared tests, and Fisher exact tests, where appropriate. Among the 147 surveyed dentists, 62.6% and 85.7% reported that they did not have any knowledge about toothbrush disinfection and did not disinfect their toothbrushes, respectively. However, approximately two thirds of surveyed dentists thought that toothbrush disinfection should be performed by everyone, including healthy individuals. Significant associations were found between knowledge about toothbrush disinfection and the professional title of dentists, how they stored their toothbrushes, and whether their toothbrushes were in contact with each other during storage (p < 0.05). A minority of dentists reported that they disinfected their toothbrushes.