Objectives: To evaluate and compare the biofilm formation between labial and lingual orthodontic brackets. Materials and Methods: Twenty patients with a mean age of 24 ± 8.8 who had received labial or lingual orthodontic treatment were enrolled in the study. Biofilm formation on 80 brackets was analyzed quantitatively with the Rutherford backscattering detection method. Five micrographs were obtained per bracket with views from the vestibule/lingual, mesial, distal, gingival, and occlusal aspects. Quantitative analysis was carried out with surface analysis software (ImageJ 1.48). Data were analyzed by Mann-Whitney U and Kruskal-Wallis tests (α = 0.05). Results: Total biofilm formation was 41.56% (min 29.43% to max 48.76%) on lingual brackets and 26.52% (min 21.61% to max 32.71%) on labial brackets. Differences between the two groups were found to be significant. No difference was observed in intraoral location. The biofilm accumulation was mostly located on gingival, mesial, and distal surfaces for both groups. Conclusions: The biofilm accumulation on lingual orthodontic therapy was found to be more than labial orthodontic therapy.