The aim of this study was to compare the antibacterial activity of fluoride varnishes containing different agents in children with severe early childhood caries (S-ECC). The study was conducted in 92 children with S-ECC. Patients who completed dental treatment under general anaesthesia were included in the study. Patients were divided randomly into four groups: 5% sodium fluoride (SF) control group (n= 23), 5% SF with tricalcium phosphate (TCP) (n= 23), 5% SF with xylitol-coated calcium and phosphate (CXP) (n= 23) and 5% SF with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (n= 23). Saliva mutans streptococci (MS) and lactobacilli (LB) levels were evaluated by taking saliva samples at baseline (T0), 1month (T1) and 3months (T2) after treatment. All groups showed a significant decrease in MS and LB levels at T1 (P< 0.05) except the CXP group. Only the TCP group exhibited significantly decrease MS and LB levels indicating less than 105CFU at both T1 and T2 compared with the other groups (P< 0.05). Fluoride varnish with TCP provided significantly more reduction in MS and LB levels than other fluoride varnishes. Fluoride varnish with TCP had a significant antibacterial effect against cariogenic bacteria until the end of the third month. Long lasting antibacterial effect provides additional advantage for reducing bacteria levels in children. NCT03625310.