Objectives: Oral bacteria implying a natural resistance may deteriorate the antibacterial efficacy of chlorhexidine on cariogenic microorganisms. Xylitol, mostly applied via chewing gum, is known to possess favorable plaque-reducing properties. The aim of this study was to investigate the effect of a xylitol rinse formulated as pure solution or combined with chlorhexidine on the viability of Streptococcus sanguis (early colonizer of human teeth) and Streptococcus mutans (the most causal strain for caries) during initial steps of biofilm formation. Method and Materials: After exposure to the test solutions, the bacteria suspended in human sterile saliva were allowed to attach to human enamel slides for 60 minutes in a preclinical flow chamber system. The bacterial vitality of suspended and attached cells was monitored using 2 fluorescent DNA stains by epifluorescence microscopy. Further parameters measured were the total bacterial cell counts on enamel slides and growth of suspended streptococci. Results: The sensitivity of S mutans to pure chlorhexidine or in combination with xylitol is contrary to the natural resistance of S sanguis to chlorhexidine. The combination of xylitol/chlorhexidine showed a statistically significant antivital effect on S sanguis cells compared to the pure agents xylitol and chlorhexidine. The bacterial cell density on enamel and bacterial reproduction on agar plates were similarly affected by the combination of xylitol/chlorhexidine or the single substances. Conclusion: The newly discovered synergistic antivital effect of xylitol combined with chlorhexidine may contribute to the favorable potential of xylitol use for the improvement of new formulations of caries-preventive mouthrinses. Keywords: chlorhexidine, initial biofilm, Streptococcus mutans, Streptococcus sanguis, synergistic effect, xylitol