The aim of this in vitro study was to determine the effects of a smear layer and chlorhexidine (CHX) treatment on the adhesion of Enterococcus faecalis to bovine dentin. Forty dentin blocks from bovine incisors were prepared and randomly divided into four groups of 10 each. The blocks in group 1 were placed in sterile saline for 5 minutes, while those in group 2 were treated with 17% EDTA for 5 minutes. The blocks in group 3 were placed in 2% CHX for 7 days. The blocks in group 4 were treated with 17% EDTA for 5 minutes, and then placed in 2% CHX for 7 days. All the blocks were immersed in a suspension of E. faecalis for 3 hours. The bacteria adhering to the dentin surface were counted by examination using a scanning electron microscope. The most significant amount of bacteria was retained on the samples from group 1 (p < 0.05) and the smallest amount of bacteria adhered to the samples from group 4. These results suggest that a smear layer enhances the adherence of E. faecalis to the dentin, and CHX is effective in reducing the adherence of microorganisms.