BACKGROUND Recent systematic reviews on clinical trials comparing the efficacy of chlorhexidine and fluoride varnish found that the evidence was inconclusive and further well-conducted randomized controlled clinical trials were advocated. OBJECTIVE To compare the effect of fluoride varnish (F) and Chlorhexidine-thymol varnish (CHX/T) with intensive application regimen on mutans streptococci (MS) levels in human dental plaque. METHODS Seventy-five subjects between 6 and 10 years of age were allocated into three groups: Group 1-F varnish (n = 29); Group 2-CHX/T varnish(n = 29); Group 3-placebo varnish (n = 17) by stratified block randomization. After baseline plaque samples were obtained, varnish application was applied and repeated at an interval of 3 days in each group. Plaque samples were repeated at 48 h, 1 month, and 3 months. The samples were spread over mitis-salivarius-bacitracin (MSB) culture media, and the colony-forming units per ml (CFU) were measured. RESULTS In both Groups 1 and 2, Wilcoxon matched-pairs signed-rank test revealed significant differences in log CFU values of MS between baseline and 48 h, baseline and 1 month but no significant difference between baseline and 3 months. An intergroup comparison at different time intervals showed that the difference between three groups was statistically insignificant. CONCLUSIONS F varnish and CHX/T varnish, with an intensive regimen application have equivocal effect on MS levels in dental plaque.