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Objectives: To identify microorganisms isolated from patients wearing fixed orthodontic appliances and to evaluate the resistance of isolated bacterial strains to different antimicrobials., Materials and Methods: Seventeen healthy patients wearing a fixed orthodontic appliance (group 1) and six nonwearers (group 2, control group) were evaluated. The biofilm that formed around the orthodontic brackets was collected, and the samples were then plated in a chromogenic medium (chromIDT, bioMérieux). Colony-forming units (CFUs) were isolated and inoculated in blood-agar medium. Automated biochemical tests (VITEK 2, bioMérieux) were carried out to identify the genus and species of the microorganisms and the resistance provided by 43 drugs (37 antibacterial and 6 antifungal)., Results: The most prevalent microbial genera identified in group 1 were Streptococcus (24.0%), Staphylococcus (20.0%), Enterobacter (12.0%), Geobacillus (12.0%), and Candida (12.0%), and the most frequent species were Enterobacter cloacae complex (13.6%) and Staphylococcus hominis (13.6%). In group 2, the most prevalent genera were Streptococcus (57.1%), Staphylococcus (14.2%), Sphingomonas (14.2%), and Enterobacter (14.2%). With regard to antimicrobial resistance, 14 of 19 (74%) isolated bacterial strains were found to be resistant to at least 1 of the tested antimicrobials., Conclusions: The findings of the present study suggest that patients undergoing orthodontic treatment with fixed appliances have a more complex biofilm with a higher level of bacterial resistance., (© 2021 by The EH Angle Education and Research Foundation, Inc.)