This quasi-experimental study aimed at investigating the combined effect of biofilm accumulation and 20% sucrose rinse on the modulation of calcium (Ca2+), phosphate (Pi), and fluoride (F-) bioavailability in saliva of children with early childhood caries (ECC). In-that, fifty-six preschoolers of both genders were evaluated according to caries experience and activity: caries-free (CF, n=28) and with ECC (n=28) and then, submitted to biofilm intervention (biofilm accumulation or no biofilm accumulation). In each situation, saliva samples were collected before and five minutes after a 20% sucrose rinse to determine the concentrations of Ca2+, Pi, and F−. Calcium concentration was significantly lower in the biofilm accumulation situation compared to the situation of biofilm mechanical control, except for children CF after sucrose rinse. Biofilm accumulation increased salivary calcium concentration in children with ECC after sucrose rinse, whereas mechanical biofilm control reduced it in both groups. The phosphate concentration was influenced by mechanical control of the biofilm in CF children. The fluoride bioavailability was reduced by sucrose rinse and biofilm accumulation in CF and ECC children. In conclusion, the combined effect of biofilm accumulation and sucrose rinse modifies the bioavailability of calcium and fluoride in the saliva of children with early childhood caries.