Purpose: The aim of the present study was to test the effects of interdental cleansing with dental floss on supragingival biofilm removal in natural dentition during a 3-week period of experimental biofilm accumulation. Materials and Methods: The present study was performed as a single-blind, parallel, randomised, controlled clinical trial using the experimental gingivitis model (Löe et al, 1965). Thirty-two students were recruited and assigned to one of the following experimental or control groups: Group A used a fluoride-containing dentifrice (NaF dentifrice) on a toothbrush for 60 s twice a day, Group B used an unwaxed dental floss twice a day, Group C used a waxed dental floss twice a day in every interproximal space and Group D rinsed twice a day for 60 s with drinking water (control). Results: During 21 days of abolished oral hygiene, the groups developed various amounts of plaque and gingivitis. Neither of the cleansing protocols alone allowed the prevention of gingivitis development. Toothbrushing alone yielded better outcomes than did any of the flossing protocols. Interdental cleansing with a waxed floss had better biofilm removal effects than with unwaxed floss. Conclusions: Toothbrushing without interdental cleansing using dental floss and interdental cleansing alone cannot prevent the development of gingivitis. Keywords: biofilm, clinical study, experimental gingivitis, inflammation, interdental cleansing, oral hygiene, plaque control