Introduction: Orthodontist must take precautionary measures to protect patients from cross-infection by contaminated materials received from the manufacturers. To minimize the incidence of cross-infection prior to bonding an invitro study was done to assess the antibacterial activity of chlorhexidine, povidone iodine and glutaraldehyde. Aim: To compare the antibacterial activity of 2% chlorhexidine, 5% povidone iodine and 2% glutaraldehyde on orthodontic materials prior to bonding. Materials and methods: A total of 36 samples were included in this  study. The main group consists of 3 disinfectants and one control group (n =4). Group -1(Chlorhexidine gluconate), Group - 2 (Povidone Iodine), Group - 3 (Glutaraldehyde) and Group - 4 (Control group –untreated). Each group consists of 3 subgroups of the tested samples namely group A (brackets), group B (archwires) and group C - molar bands (n=9). which were contaminated by 3 bacterial species streptococcus, staphylococcus, lactobacillus and the number of colonies were counted before disinfection. The contaminated samples were subjected to disinfection with 2% chlorhexidine for 5 minutes 2% glutaraldehyde and 5% Povidone Iodine for 10 minutes. 20µl of sample of the disinfected samples were incubated and the number of colonies were counted after disinfection. Results: There were no colonies formed when 5% Povidone iodine was used to disinfect archwires and molarbands in few tested samples. 2% glutaraldehyde and 2% chlorhexidine were not effective against lactobacillus species. Povidone iodine was effective when compared to glutaraldehyde and chlorhexidine gluconate and effective against all the three species namely Streptococcus, Staphylococcus and Lactobacilli species. The obtained p-value is < 0.001 which is statistically significant when all groups were compared overall. Conclusion: Disinfection with 5% povidone iodine was most effective when compared with 2% chlorhexidine and 2% glutaraldehyde.

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