Purpose: This double-blind, clinical, cross-over study evaluated the antibacterial effect of three toothpastes (ASF, HTP and STP) and a chlorhexidine mouthrinse (0.2%; CHX; positive control) after a single application on established biofilm over a period of 24 h (substantivity). Materials and Methods: Twenty-four subjects refrained from all oral hygiene measures for a period of 72 h. After 48 h, a baseline biofilm sample was taken and vitality of the biofilm flora was examined (baseline, VF0). Then they rinsed for 1 min with one of the randomly allocated, freshly prepared toothpaste slurries (ASF, HTP, STP) or CHX. Further biofilm samples were taken every second hour up to 14 h as well as 24 h after rinsing, and biofilm vitality was assessed (VF2-24). After a wash-out period of 4 days, a new test cycle was started. Results: All subjects (18 female, 6 male) finished the four test cycles. At VF2, all products showed a statistically significant reduction in vitality compared to VF0 (p0.05). CHX and ASF revealed the most pronounced effect (49% and 40% reduction), while the other toothpastes (HTP: 24%, STP: 11%) reached lower but still statistically significant effects. At each further time point CHX and ASF showed the lowest biofilm vitality. ASF demonstrated a significant antibacterial effect on dental biofilm over a 24-h period compared to baseline and superiority over both other toothpastes at time points VF2-VF14. Conclusion: ASF toothpaste showed a significant antibacterial action on biofilm and a high substantivity which was maintained up to 24 hours. Keywords: amine fluoride, biofilm vitality, chlorhexidine, substantivity, stannous fluoride, toothpaste