In recent years, a new endodontic cement, calcium enriched mixture (CEM) has been introduced; with clinical applications similar to those of mineral trioxide aggregate (MTA). It has been shown that CEM has higher antibacterial activity than MTA. On the other hand, use of chlorhexidine (CHX) to promote the antibacterial activity of different dental materials is increasing. The aim of the present study was to evaluate the effect of adding CHX to CEM on its antibacterial activity. The antibacterial activities of the materials under study [(CEM cement + CEM solution + 2%CHX) and (CEM cement + CEM solution)] against Pseudomonas aeroginosa, Enterococcus faecalis, Staphylococcus aureus and Escherichia coli were evaluated using agar diffusion technique, followed by determination of the diameter of microbial zone of inhibition around the materials by three independent observers after 72 h. Data were analyzed by Mann-Whitney U test. Statistical significance was defined at P<0.05. The mean diameters of zones of inhibition in the CEM + CEM liquid and CEM + CEM liquid + CHX groups against P. aeroginosa, E. faecalis, S. aureus and E. coli were (13.2 and 9), (21.10 and 6), (20.2 and 9) and (17 and 9.75) ml, respectively, with larger diameters in the CEM + CEM solution + CHX group as compared to CEM + CEM solution group with all the microorganisms (P<0.05). Incorporation of CHX with CEM resulted in an increase in antimicrobial activity of CEM.