BACKGROUND Various strategies for controlling caries focus on disrupting the interaction between risk factors. Of these, fluoride varnish has been shown to reduce the colony-forming (CFU) units and water-insoluble extracellular polysaccharide amount. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing fluoride varnishes have recently gained importance as caries-protective fluoride varnishes. OBJECTIVE This study aims to assess and compare the reduction in Streptococcus mutans count in biofilm samples after topical application of three different fluoride varnishes and to evaluate the effect of oral prophylaxis prior to fluoride varnish application. METHODS Sixty healthy children with no active caries, in the age group of 2-8 years, were randomly divided into Group A = fluoride varnish containing CPP-ACP; Group B = fluoride varnish containing xylitol; and Group C = fluoride varnish with 0.9% difluorosilane; further, the groups were divided into two subgroups, namely A1, B1, and C1 with prior oral prophylaxis and A2, B2, and C2 without oral prophylaxis. Plaque samples were collected at baseline, 1st month, and 3rd month; cultured; and incubated, and CFU/ml was calculated. RESULTS Data were compiled, and CFU/ml was analyzed by independent t-test, paired t-test, and one-way ANOVA. There was no statistical difference between the fluoride groups. Furthermore, no statistically significant difference was seen between the subgroups. CONCLUSIONS Fluoride varnish containing CPP-ACP showed higher reduction in S. mutans count followed by xylitol-containing fluoride varnish and Fluor Protector®. There was no effect of prior oral prophylaxis on the efficacy of fluoride varnish.