Purpose: The purpose of this study was to evaluate the effect of whole bovine milk on Streptococcus mutans biofilms and on demineralization of enamel and root dentin. Methods: S mutans UA159 biofilms were cultured on enamel and dentin bovine slabs of known surface hardness (SH) and exposed (8x/day) to 10% sucrose (positive control), whole bovine milk, and 4.5% lactose. Biofilm acidogenicity was estimated by medium pH (2x/day). After 4 days for dentin and 5 days for enamel, biofilms were collected to obtain biomass, viable bacteria, total protein, and intra- and extracellular polysaccharides. Final SH was measured on the slabs. Demineralization was calculated as percentage of SH loss (%SHL). Differences were determined by analysis of variance and Tukey's test (P<.05). Results: Biofilms formed on both substrates under 10% sucrose showed higher acidogenicity, biomass, protein concentration, insoluble polysaccharides, and %SHL than those treated with milk or lactose (P<.05), which did not differ in any parameter (P>.05). Higher bacterial counts were obtained from biofilms treated with milk on enamel, but not on dentin. Dentin was significantly more demineralized by milk than enamel (P<.05). Conclusions: Although bovine milk appears to be less cariogenic than sucrose, this dairy product should not be considered caries-safe.