It was reported that clinical application of chlorhexidine (CHX) varnish could reduce dental caries occurrence effectively. However, this form of CHX is not commercially available in Thailand. Our previous study showed that the released CHX from 20% and 40% self-prepared CHX varnishes were sufficient to inhibit the growth of Streptococcus mutans. This study aimed to compare the efficacy of the 20% and 40% self-prepared CHX varnishes to commercial CHX varnish EC 40®. The study included CHX release, antibacterial activity against S. mutans ATCC 25175, and the cytotoxic effect of CHX on fibroblasts. The results showed that the greatest amount of CHX was released by EC 40® followed by 40% and 20% selfprepared CHX, which were 4,111.29 μg, 2,408.7 μg, and 1,136.4 μg, respectively. EC 40® gave the strongest antibacterial activity; however, there was no statistical significant difference. The 20% self-prepared CHX gave the highest viability of fibroblasts. This study indicates that the self-prepared CHX should be considered to be used as antimicrobial agents for the prevention of dental caries.