Purpose: This study aimed to assess the efficacy of palatal brushing in the treatment of denture stomatitis. Materials and Methods: After screening 143 individuals with a potential diagnosis of denture stomatitis, 48 patients (mean age: 66.0 ± 11.2 years) were enrolled in a two-center phase 1 clinical trial with a one-group pretest/posttest design. The intervention of interest was manual palatal brushing after each meal and before bedtime. Clinical and microbiologic examinations were performed at baseline and 1 and 3 months after treatment. Additional data were obtained using a validated questionnaire. The primary and secondary outcomes were the remission of denture stomatitis and diminution of Candida colony-forming units (CFUs), respectively. Descriptive and nonparametric statistical tests were conducted to analyze the data. Results: At the 3-month follow-up, denture stomatitis was completely cured in 10.4% of the participants, and 70.8% of denture wearers showed improvement in the clinical signs of denture stomatitis. There was a significant reduction in the area and severity of the palatal inflammation (P .0001). The effect size ranged from medium to large (0.34 to 0.54) depending on the classification used for the diagnosis of denture stomatitis. A significant reduction in the number of Candida CFUs isolated from the palatal mucosa and dentures (P ≤ .05) was observed. Conclusions: The results of this study suggest that palatal brushing is an effective treatment of denture stomatitis.