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Introduction. Periodontitis is among the most widespread oral bacterial diseases affecting 15-20% of the world population. Aim . This study aimed to develop dental floss impregnated with povidone-iodine (PVP-I) as an antimicrobial delivery system against periodontopathogenic bacteria in a planktonic form and within biofilms. Methods. Identical lengths of dental floss impregnated with PVP-I formulations were placed on agar along with previously grown periodontal pathogens. The bioactivity of the dental floss was investigated by response-surface methodology. In order to explore the antibacterial activity of the selected formulation and the potential application in the prevention and treatment of plaque-caused diseases such as periodontitis and caries, the antibacterial and anti-biofilm activity of the selected PVP-I formulation against pathogenic bacteria were investigated. Results. The results indicated that the coating formulation containing Eudragit L-100 2.90 %, PVP-I 24.58 % and PEG 400 3.73 % had antimicrobial activity for all pathogens. The mechanism of this formulation involved disruption of bacterial cell membranes. Moreover, this formulation inhibited the formation of oral pathogenic biofilms. Conclusion. It was concluded that Eudragit L-100 and PVP-I-coated dental floss represented a potential therapeutic agent to prevent periodontal diseases and dental caries and exhibited non-toxicity to periodontal ligament cells.