Iron (Fe) may have an anticaries effect by specific inhibition of glycosyltransferase (GTF) enzymes of Streptococcus mutans, but this hypothesis has not yet been clarified. In this study, S. mutans biofilms were formed on blocks of bovine dental enamel of a predetermined surface hardness (SH). These biofilms were exposed eight times/day to 10% sucrose, and two times/day they were subjected to one of the following treatments: G1, 0.9% NaCl as a negative control; G2, 0.12% chlorhexidine digluconate (CHX) as a positive antibacterial control; G3, 0.05% NaF (225 ppm F) as a positive anticaries control; G4, G5, and G6, ferrous sulfate (Fe2+) at concentrations of 1.0, 10.0, and 100.0 µg Fe/mL, respectively. The experiment was performed in triplicate and was repeated three times (n = 9). The pH of the culture medium was determined every 24 h as an indicator of the biofilm's acidogenicity. The biofilm formed on each block was collected for determination of the viable bacteria and concentration of extracellular polysaccharides (EPS). Enamel SH was again determined and the percentage of SH loss (%SHL) was calculated as an indicator of demineralization. Iron treatment reduced the number of viable bacteria formed in the S. mutans biofilm (p = 0.04), in a dose-dependent manner, and also reduced the enamel's %SHL (p = 0.005). At 100 µg/mL, Fe reduced enamel demineralization as effectively as CHX and NaF (p < 0.05), but it did not inhibit EPS production. In conclusion, the data suggest that the anticaries mechanism of action of Fe may not involve the oxidative inhibition of GTFs.