We aimed to assess the antimicrobial efficacy of four calcium hydroxide formulations and chlorhexidine gel against E. faecalis in vitro. We evaluated the effect of CHX gel and calcium hydroxide in combination with various vehicles using the agar diffusion test to measure the zone of inhibition. In the agar diffusion test, 1% chlorhexidine gluconate gel showed better antibacterial efficacy against E. faecalis. Vitapex paste with 30% Ca (OH)2 showed the least inhibition of E. faecalis. CHX was found to be effective against E. faecalis in vitro. Further in vivo studies are needed to confirm the value of CHX in clinical treatment.