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Background: Soft denture lining-materials are more susceptible to microbial adhesion than hard denture base acrylic resin. Poor oral hygiene and Candida albicans infection are common among elderly denture wearers as these patients usually have difficulty in keeping them clean., Purpose: To evaluate the influence of the oral hygiene methods on the formation of a biofilm over a soft denture-lining material., Material and Methods: Twenty volunteers were randomly separated into two groups: G1 and G2. Ten volunteers performed daily hygiene of the prostheses with a soft toothbrush and toothpaste. The G2 performed a treatment identical to G1 but also immersed the prostheses in sodium hypochlorite 0.5% for 20 min, once a week. Quantification of the mean score values of biofilm formation at different times were statistically analysed using analysis of variance and Tukey's test (alpha = 0.05)., Results: G1 (0.65 +/- 0.52) showed the lowest mean score values of biofilm formation. There was statistical difference between G1 and G2. The highest mean score values were found at 6 weeks (1.3 +/- 1.08) and were statistically different from other times., Conclusion: The oral hygiene methods had a significant effect in the formation of the biofilm over a soft denture-lining material.