Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome. Periodontitis, as chronic inflammatory destructive disease, is associated metabolic syndromes bidirectionally. Toothbrushing is an essential and important way to manage periodontitis through mechanical removal of biofilm at periodontal tissue. We aimed to assess the association between toothbrushing frequency and the prevalent NAFLD in nationally representative Korean adults. Among adults aged 19 years and older who participated in the Korea National Health and Nutrition Examination Survey in 2010, a total of 6,352 subjects were analyzed. NAFLD was defined as fatty liver index ≥60. Multiple logistic regression analysis was used to estimate multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs). An inverse association between toothbrushing frequency and NAFLD was found. The adjusted ORs (95% CIs) of NALFD was 0.56 (0.35-0.91) in the group who performed toothbrushing ≥ 3 per day compared to the group that performed toothbrushing ≤ 1 per day. For those with toothbrushing frequency ≤1 per day, the adjusted OR (95% CIs) of NAFLD was 2.26 (1.22-4.19) in smokers and 4.52 (1.97-10.38) in subjects with diabetes mellitus (DM), compared to those without the disease and with toothbrushing frequency ≥2 per day, respectively. Our results indicate that higher frequency of toothbrushing is inversely associated with NAFLD. As a modifiable oral habit, regular toothbrushing may be recommended to lower risk of NAFLD, especially in high risk groups such as smokers and diabetic patients.