A previous study demonstrated that alexidine has greater affinity for the major virulence factors of bacteria than chlorhexidine. The aim of this study was to compare the antimicrobial activity of 1% alexidine with that of 2% chlorhexidine using Enterococcus faecalis-infected dentin blocks. Sixty bovine dentin blocks were prepared and randomly divided into six groups of 10 each. E. faecalis was inoculated on 60 dentin blocks using the Luppens apparatus for 24 h and then the dentin blocks were soaked in 2% chlorhexidine or 1% alexidine solutions for 5 and 10 min, respectively. Sterile saline was used as a control. The antimicrobial efficacy was assessed by counting the number of bacteria adhering to the dentin surface and observing the degradation of bacterial shape or membrane rupture under a scanning electron microscope. Significantly fewer bacteria were observed in the 2% chlorhexidine- or 1% alexidine-soaked groups than in the control group (P<0.05). However, there was no significant difference in the number of bacteria adhering to the dentinal surface between the two experimental groups or between the two soaking time groups (P>0.05). Ruptured or antiseptic-attached bacteria were more frequently observed in the 10-min-soaked chlorhexidine and alexidine groups than in the 5-min-soaked chlorhexidine and alexidine groups. In conclusion, 10-min soaking with 1% alexidine or 2% chlorhexidine can be effective against E. faecalis infection.