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Abstract Background Contamination of orthodontic appliances is due to the unhygienic practices followed during manufacturing and packaging processes, which may lead to cross-contamination. Although literature has indicated the need for sterilization or disinfection of orthodontic appliances before using in the oral cavity, this is still not employed in routine clinical practice. In this view, the current study evaluates the bacterial load on orthodontic brackets along with the disinfecting efficacy of chlorhexidine. Methods A total of 140 brackets were obtained from four different manufacturers and divided into six groups: group 1 (American Orthodontics; n = 30), group 2 (3M Unitek; n = 30), group 3 (Ortho Organizers; n = 30), group 4 (China Dental Orthodontic; n = 30), group 5 (negative control; n = 10), and group 6 (positive control; n = 10). Various microbiological and biochemical tests were conducted on the brackets to detect the type and growth of bacteria. Brackets that showed microbial contamination were then subjected to disinfection using 0.01% and 2% chlorhexidine solutions. Results Microbial contamination was detected on brackets of all the four groups. Bacteria, including Staphylococcus aureus, S. epidermidis, Lactobacilli, Klebsiella pneumoniae, Bacillus licheniformis, and B. cereus, were identified in these groups. Upon disinfection with 0.01% chlorhexidine solution, brackets in group 2 displayed complete decontamination, while all brackets in the other groups containing gram-negative bacteria exhibited complete decontamination with 2% chlorhexidine. Conclusion Orthodontic brackets received from four manufacturers showed high bacterial contamination. Disinfecting ability of 2% chlorhexidine proved highly effective in destroying both gram-positive and gram-negative bacteria. Therefore, use of 2% chlorhexidine in clinical practice for the disinfection of orthodontic brackets is suggested, before placement in the oral cavity.