The aim of this study was to quantify biofilm on the internal surface of upper complete dentures following six possible cleansing methods. Thirty-six edentulous subjects were submitted to a time-series trial and dentures were cleansed according to six methods: (i) rinsing with water; (ii) soaking in an alkaline peroxide solution (Bonyplus); (iii) brushing with dentifrice (Dentu-Creme) and soft Johnson and Johnson's toothbrush; (iv) combination between soaking and brushing according to methods 2 and 3; (v) brushing with dentifrice (Dentu-Creme) and soft Oral B toothbrush; (vi) combination between soaking and brushing according to methods 2 and 5. Each method was randomly used throughout 21 days. Denture biofilm was disclosed by 1% neutral red solution and quantified by means of digital photos taken from the internal surface. The six methods presented significant differences in percentage of biofilm coverage (repeated measures anova, P < 0.0001). Method 1 showed the highest values, 2 was intermediate and other results were the lowest. The most efficacious approach was 6. Biofilm tended to accumulate predominantly over specific zones of the denture base, but this pattern did not change regardless of the method employed. It can be concluded that brushing alone was more effective than the chemical method employed. The best results were obtained by a combination of methods.