The intention of this study was to investigate the preventive effect of chlorhexidine varnish on enamel demineralization. The sample consisted of 57 first premolars scheduled to be extracted for orthodontic purposes. The control group (N=10) was left untreated and the experimental groups were worn with specially designed stainless steel orthodontic bands on premolar for plaque accumulation. The group 1 (N=9) was worn band only, the group 2 (N=19) was applied with chlorhexidine varnish for one time, and the group 3 (N=19) was applied with chlorhexidine varnish for 3 times once a week. After 4 weeks of experimental periods, every specimen were examined by SEM and Vickers hardness test to evaluate and compare the degree of enamel decalcification. The results were as follows: 1. Although SEM revealed various degree of enamel demineralization in every experimental groups, the group 1 showed more severe demineralizations than the group 2 and 3. 2. The mean Vickers Hardness Numbers measured in this study seemed to reveal that there was a statistically significant difference between the control goup and the group 1 (P<0.05), and also a significant difference between the group 1 and the group 2, 3 (P<0.05). And there was no significant difference between the group 2 and the group 3 (p>0.05). 3. The results of VHN did not deemed to show a statistically significant difference between maxillary premolar and mandibular premolar in both group 2 and group 3 (P>0.05).