Background: Dental caries continues to be a major public health problem in many countries. The etiology of dental caries is multifactorial, and it is widely accepted that Mutans streptococci and Lactobacilli play a major role in the formation of dental caries through adhering to the tooth surface and producing acid from dietary sucrose. Chlorhexidine is active against both gram positive and negative bacteria and Xylitol is a naturally occurring non-cariogenic sugar substitute used as an artificial sweetener in foods, Objective: The purpose of this study was to determine the impact of xylitol on the antibacterial activity of chlorhexidine against salivary Mutans streptococci and lactobacilli. Results: To evaluate the effects of these substances salivary mutans streptococci and lactobacilli were isolated and identified, Agar diffusion technique was used to study the antimicrobial effects of both xylitol and chlorhexidine. The minimum inhibitory concentration was determined for the xylitol and chlorhexidine alone and in combination using tube dilutions method, the combination effects of xylitol and chlorhexidine were evaluated using fractional inhibitory concentration (FIC) indices. Conclusion: ½ MIC of chlorhexidine with ¼ MIC of xylitol was efficient and superior to single treatments in controlling suppressing mutans streptococci and lactobacilli in-vitro and xylitol may be used as sweetener and flavoring chlorhexidine.