Objective: To investigate the effect of silver fluoride in preventing the development of artificial caries lesions in root dentine using an artificial mouth system (AMS). Methods: A total of 34 extracted intact human premolars were embedded individually in blocks with one root dentine surface exposed. Among these, 32 were randomly divided into four groups (eight each) and the remaining two tooth blocks were used in the baseline evaluation of the cariogenic biofilm after bacterial inoculation. The interventions (topical application of 2.36 M solutions) were applied after inoculation as follows: group-1, silver fluoride (AgF); group-2, potassium fluoride (KF); group-3, silver nitrate (AgNO3); and group-4, deionised water (control). Subsequently, the tooth blocks were transferred into the AMS to start an artificial caries challenge (5% sucrose was delivered three times per day). After 10 days, the formed biofilm was assessed via colony forming unit (CFU) counts, confocal laser scanning microscopy, and scanning electron microscopy. The artificial dentinal caries lesion was evaluated using micro-computed tomography. Results: After the interventions and the 10-day challenge in the AMS, the median CFUs (AgF AgNO3 KF = control, P 0.001) and the median live-to-dead bacteria ratios (AgF AgNO3 KF = control, P 0.005) of the biofilm differentiated significantly among the groups. Scattered bacterial cells were found in the tooth blocks of the AgF and AgNO3 groups, while a confluent biofilm layer was observed in the tooth blocks of the KF and control groups. The median lesion depth in the AgF group was significantly lower than in the AgNO3 (P = 0.016), KF (P = 0.016) and control (P = 0.009) groups. Conclusion: The combined use of silver and fluoride ions in the AgF solution significantly protected dentine against the development of caries lesions. Keywords: silver fluoride, dental biofilm, artificial mouth system, dentine caries lesion, prevention