Aim: The aim of this randomized clinical trial was to evaluate the bacterial survival rate on toothbrushes and efficacy of their decontamination by4% disodium ethyl diamine acetic acid [EDTA], 10% sodium perborate and compared with control. Methods: Thirty subjects with chronic periodontitis enrolled in this randomized controlled clinical trial were provided with autoclaved toothbrushes which were free from microorganisms. Brushing instructions were given to each participant. Toothbrushes were collected from all study participants after 1 week and were placed with head down position in an autoclaved test tube containing sterile peptone water. Toothbrushes collected were sent for aerobic culture in laboratory for growth of micro-organisms. Incubation was done for 24 hours at 370C.The toothbrushes were then divided into three groups and immersed in disinfectantslike4% disodium EDTA, 10% sodium perborate and their efficacy was evaluated by aerobic culture analysis. Chi – Square test was used for statistical analysis of the data. Results: Escherichia coli, Pseudomonas Aeroginosa, Streptococci and Klebsiella species were recovered from the samples. The results obtained showed that 4% Disodium EDTA showed 100% efficacy, whereas 10% Sodium perborate showed 40% effectiveness in decontaminating the toothbrushes. Distilled water as a control showed least effectiveness in cleaning toothbrushes. Conclusion: After single brushing toothbrushes get contaminated by a wide array of bacteria’s which a major cause of concern is. As contaminated toothbrush can reintroduce microorganisms into the oral cavity, it is therefore recommended for individuals to use solutions like 4% Disodium EDTA, which proved to be an effective disinfecting agent for decontaminating toothbrushes.