Objectives: This in vitro study investigated the effects of silver diamine fluoride (SDF) on dentine carious lesion with cariogenic biofilm. Study Design: Thirty human dentine blocks were inoculated with Streptococcus mutans and Lactobacillus acidophilus dual-species biofilm to create carious lesion. They were equally divided into test and control group to receive topical application of SDF and water. After incubation anaerobically using micro-well plate at 37oC for 7 days, the biofilms were evaluated for kinetics, morphology and viability by colony forming units (CFU), scanning electron microscopy (SEM), and confocal microscopy (CLSM), respectively. The carious lesion underwent crystal characteristics analysis, evaluation of the changes in chemical structure and density of collagen fibrils using x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and immune-labeling. Results: The log CFU of S. mutans and L. acidophilus in the test group was significantly lower than control group. SEM and CLSM showed confluent biofilm in control group, but not in test group. XRD showed the loss of crystallinity of dentine due to the dissolution of hydroxyapatite crystal structure in test group was less than control group. FTIR showed that log [Amide I: HPO4 2-] for test vs. control group was 0.31±0.10 vs. 0.57±0.13 (p<0.05). The goldlabeling density in test vs. control group was 8.54±2.44/ìm2 vs. 12.91±4.24/ìm2 (p=0.04). Conclusions: SDF had antimicrobial activity against the cariogenic biofilms and reduced demineralization of dentine.