ABSTRACT: Periodontal disease is clearly correlated with systemic disease. The presence of periodontal pathogens in interdental spaces in young, healthy adults is a strong indicator of the need to introduce daily interdental prophylaxis. Twenty-five subjects (aged 18-35 years), diagnosticated clinically as periodontally healthy, were enrolled in this study. One hundred interdental sites were included. Among these sites, 50 "test" sites were cleaned daily with calibrated interdental brushes (IDBs), whereas the other 50 sites were not cleaned and considered "controls". The interdental biofilm at these interdental sites was collected at the beginning of the study (basal) and at 1 week, 2 weeks, 3 weeks, 4 weeks, and 3 months. Real-time polymerase chain reaction (PCR) methodology was used to quantify (i) 19 periodontal bacteria, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, and (ii) total bacteria. In the test sites, the quantity of total bacteria decreased over time with the use of IDBs. The bacteria from the red and orange Socransky complexes, which are associated with periodontal disease, significantly decreased in the test sites but not in the control sites. Bacteria from the yellow, and purple Socransky complexes, which are associated with periodontal health, increased significantly in both groups whereas bacteria from the blue Socransky complex increased significantly only in the test sites. Furthermore, at basal, 66% of test sites and 68% of control sites bled during interdental brushing. These percentages decreased by 85% in 3 months for the test sites and by 27% in the control sites. In conclusion, the daily use of calibrated IDBs can reduce periodontal pathogens, reestablish symbiotic microbiota and, decrease interdental inflammation in interdental sites of healthy young adults.