Objectives: To study the preventive effects of chlorhexidine against root caries under oral biofilm in an artificial mouth. Study Design: Sixteen human tooth-root disks were inoculated with a salivary sample that was produced by mixing the unstimulated saliva of three adults who had no untreated caries. The disks were incubated in an artificial mouth fed with a 5% sucrose solution three times daily for one week. Eight disks received a twice daily rinse of 0.12% chlorhexidine (test group). The other eight disks were rinsed in distilled water (control). The biofilm was then studied with three techniques: colony forming unit (CFU) counting, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The changes in the chemical structure of the root surface were studied using Fourier transform infra-Red spectroscopy. Type-I collagen and proteoglycans on the root surface were quantified using immunocytochemical staining. Results: The log CFU for the test and control groups were 4.21 and 8.27, respectively (p<0.001). The CFU count of Streptococci and Lactobacilli were negligible. Both the SEM and the CLSM showed suppressed bacteria growth in the test group. The log [amide-I: HPO 4 2-] of the test and control groups were 1.11 and 1.93, respectively (p=0.02). The mean counts of sound type-I collagen in the test and control groups were 16.8/µm² and 13.0/µm², respectively (p<0.001), whereas the mean counts of intact proteoglycans were 5.6/µm² and 3.5/µm², respectively (P<0.001). Conclusions: Chlorhexidine suppressed the growth of selected cariogenic bacteria in oral biofilm on the root surface and thus protected tooth-root from cariogenic challenge.