There is growing concern about cross infection among the patients to patients, patients to staffs, and tools to patients in healthcare facilities, especially in dentistry. In this study, the most widely used dental impression materials were prepared and the synergy effect of Chlorhexidine and essential oil on antimicrobial activity was examined in the impression materials. Chlorhexidine concentration of 0.1 wt% and 0.5 wt% showed no antimicrobial activity on Escherichia coli (E. coli) and Candida albicans. At 1.0 wt% Chlorhexidine, 0% of E. coli and 34.7% of Candida albicans were survived. Bergamot (Essential oil) concentration of 0.5 wt% and 1.0 wt% showed no antimicrobial activity on E. coli. At 2.0 wt% Bergamot oil, 71.9% of E. coli were survived. Tea tree oil (Essential oil) of 0.5 wt% showed no antimicrobial activity on E. coli. At 1.0 wt% Tea tree oil, 11.2% of E. coli was survived. At 2.0 wt% Tea tree oil, no E. coli was survived. However, no E. coli was survived at the concentration of 0.8 wt% Bergamot with 0.3 wt% Chlorhexidine. At the concentration of 0.8 wt% Tea Tree oil with 0.3 wt% Chlorhexidine, 1.3% of E. coli were survived. The experimental results showed that the synergy effects between Chlorhexidine and essential oils on antimicrobial activity were prominent.