Chlorhexidine, the most used biocide in periodontology, alters the permeability of the bacterial cell membrane. However, the chlorhexidine-induced morphological alterations in the oral biofilm have not been studied. To examine the effects of chlorhexidine on oral biofilm on an electron microscopic level, gingival epithelial cells with attached biofilm were collected from 10 volunteers, subjected to 0.1% chlorhexidine for 1 or 5 min, stained with ruthenium red-tetroxide, and analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM visualized the bacterial glycocalyces and the biofilm matrix on the biofilm surface; however, no chlorhexidine-induced alterations were observed. TEM revealed loss of bacterial membrane integrity and fimbrial disintegration in a few bacteria. In the proximity of these alterations, a restricted matrix disintegration was also observed. However, the chlorhexidine-induced alterations only effected a minor part of the oral biofilm and did not cause its disintegration. These findings suggest the insufficient efficiency of chlorhexidine against oral biofilm.