Studies assessing the long-term effects of brushing with cleaning and disinfecting agents on surface roughness and biofilm accumulation on the three-dimensionally (3D) printed resins remain scarce. This study aimed to evaluate the effects of different solutions and simulated brushing times on the surface roughness and biofilm formation on heat-cured and 3D-printed denture base resins. Discs samples (10 mm × 1.2 mm) were prepared and randomly assigned to brushing treatments using the following solutions: distilled water, disinfectant liquid soap and dentifrice. The specimens (n = 9) were subjected to 10,000, 20,000 and 50,000 cycles to simulate 1, 2 and 5 years of brushing, respectively. The control group consisted of unbrushed samples. Surface roughness (Ra) was measured. Candida albicans biofilm formation was evaluated by counting colony forming units, cellular metabolic activity, and qualitative and quantitative analysis through confocal fluorescence microscopy. A significant increase in roughness was observed in both resins after two years of simulated brushing, mainly with dentifrice. After two years of brushing, an increase in the number of cells and metabolism of C. albicans was observed, in agreement with the fluorescence and biofilm thickness results. Brushing with dentifrice increased the roughness of heat-cured and 3D-printed resins and potentially increased C. albicans biofilm formation.