Background: A toothbrush is one of the most important aids used for cleaning teeth by most people in the world. So, knowledge regarding toothbrush disinfection is of utmost importance to everyone, especially the dental fraternity. With this in mind this study aims to understand the Knowledge Attitude and Practice of dentists regarding toothbrush hygiene and disinfection in private dental colleges of Lucknow City. Materials and methods: This was a cross-sectional questionnaire-based study, which included the undergraduates (UGs, interns), postgraduates (PGs), and faculty members of the four private dental colleges who gave the consent to participate in the study. The statistical analysis was done using SPSS version 22, the Chi-square test was used and p-value <0.05 was considered statistically significant. Results: There was a statistically significant difference regarding toothbrush contamination, mode of storing the toothbrush, and the various practice used for toothbrush disinfection among the interns, postgraduates, and faculty members who participated in the study. It was also seen that the general awareness and practice of toothbrush disinfection was higher among the female participants in the study, which was also a statistically significant result. Conclusion: There was a statistically significant difference in the perceptions about toothbrush contamination and disinfection among postgraduates and interns that might be attributed to their higher academic knowledge and clinical experience.