The prevalence of root-surface caries is increasing. We hypothesized that some restorative materials are protective against cariogenic challenge on root surfaces. Our goal was to study the effects of different restorative materials on root surfaces incubated with an oral biofilm generated in an artificial mouth. A biofilm of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, and Actinomyces naeslundii was co-cultured for 21 days on 24 glass-ionomer cement, resin-modified glass-ionomer cement, or resin-composite-restored root surfaces. These surfaces were then examined with Fourier transform infrared spectroscopy and scanning electron energy-dispersive spectroscopy. Only glass-ionomer restorations showed a significant increase in log calcium-to-phosphorus ratio (P < 0.01), and a significantly lower log amide I-to-hydrogen phosphate ratio on the root surface after incubation in the artificial mouth. Glass-ionomer restoratives conferred a preventive effect on the root surfaces against initial cariogenic challenge with a mixed-species oral biofilm without therapeutic intervention.