Background: With the increasing frequency of root caries, remineralization of caries lesions has become very important. Objective: To compare the effects of different fluoride varnishes on artificial root caries lesions and biofilm formation. Materials and Methods: Bovine root dentin specimens (n=138) were prepared and demineralized for five days. Dentin blocks were randomly allocated to six groups (n=23/group): (1) untreated control; (2) Sodium Fluoride (NaF); (3) NaF+ Casein Phosphopeptide-stabilized Amorphous Calcium Phosphate complexes (CPP-ACP); (4) NaF+ Tricalcium Phosphate (TCP); (5) NaF+ Xylitol-coated Calcium and Phosphate (CXP); and (6) Silver Diamine Fluoride/Potassium Iodide (SDF/KI). Specimens spared for spectral analysis (n=15) were subjected to pH cycling for eight days. Before and after pH cycling, FTIR-ATR and laser fluorescence were used to detect changes in organic/inorganic content. S. mutans and L. casei dual-species biofilm were performed on artificial caries lesions treated with remineralization agents (n=8). Bacterial biofilm formation was evaluated with CLSM and SEM. Results: According to the laser fluorescence results, SDF/KI showed maximum remineralization values after pH cycling. All treatments reduced LF values significantly compared with control group. FTIR-ATR showed chemical reactions within dentin tissue in SDF/KI group. A significant difference in decrease of hydrogen phosphate-to-amide I ratio was observed mostly in the control group, in contrast to the decrease observed in the other four test groups. CLSM analysis showed that all varnishes are effective on total and viable biomass. However, NaF was the most effective material preventing biofilm formation. Conclusion: The application of varnishes reduced biofilm formation and demineralization of root caries.