Objective: The presence of a biofilm is necessary for both initiation and progression of dental caries. Silver-based preparations incorporated into, or applied onto, various materials designed for medical use have been shown to be effective in inhibiting biofilm formation. The purpose of this in vitro study was to measure whether a topical application of diamine silver fluoride (AgF) followed by potassium iodide (KI) on partially demineralized dentin affected the formation of a Streptococcus mutans biofilm. Method and Materials: Forty partially demineralized dentin disks were divided into 4 groups as follows: 10 disks as a control, 10 disks treated with AgF followed by KI, 10 disks treated with KI, and 10 disks treated with AgF. The outer surfaces of the disks were examined with a scanning electron microscope. Cross sections of the disks were subjected to electron probe microanalysis (EPMA) to determine the levels of calcium, phosphorous, silver, and fluoride in the dentin. Results: An S mutans biofilm covered the entire exposed surfaces of all control and KI-treated disks. No discernible bacterial biofilm was detected on disks treated with AgF or AgF/KI. Detectable amounts of silver and fluoride were found up to 450 µm in the AgF and AgF/KI sections. Conclusions: Demineralized dentin disks treated with AgF and AgF/KI prevented the formation of an S mutans biofilm and were significantly more resistant to further demineralization than the control and KI-treated disks over the experimental period. The presence of silver and fluoride in the outer layers of the disks treated with AgF and AgF/KI was the likely cause of the prevention of biofilm formation. Additional studies are required before any clinical recommendations can be made. Keywords: biofilm, demineralized dentin, potassium iodide, silver fluoride, Streptococcus mutans