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Background and Objectives: Dental caries is an infectious transmissible disease resulting from tooth-adherent specific bacteria, primarily Mutans streptococci. Fluoride varnish is an effective antimicrobial agent against Streptococcus mutans (S. mutans). The aim of the study was to evaluate the levels of S. mutans in children following application of a fluoride varnish containing CPP-ACP in mother–child pairs. Materials and Methods: Sixty children aged 18–36 months, at high caries risk along with their mothers formed the study group. Baseline levels of S. mutans in saliva of mothers and dental plaque of children was microbiologically assessed for S. mutans using the culture method. They were divided into three groups, consisting of 20 mother–child pairs. Group 1: Application of varnish to mother and saline as a placebo to child; Group 2: Applying varnish to child and saline as a placebo to mother; Group 3: Applying varnish to both mother and child. S. mutans levels in dental plaque of children were assessed at 3, 6, 9, and 12 months. Data obtained were subjected to statistical analysis using one way analysis of variance, paired t-test and multiple Post hoc tukey. The level of Significance was considered at 5% Results: In all the groups, there was a significant reduction of S. mutans levels in dental plaque of children at 3, 6, 9, and 12 months. The highest reduction in S. mutans was in Group 3 (95.33%) followed by (95.12%) in Group 2 and 40.6% in Group 1 (P < 0.05). Conclusion: Application of fluoride varnish containing CPP-ACP to the children gave equivocally significant reduction in S. mutans levels of dental plaque in children, as compared to applying in both mother and child.