This 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 the saliva of children with early childhood caries (ECC). 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). 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 (p ≤ 0.01), except for CF children after sucrose rinse. Biofilm accumulation increased salivary calcium concentration in children with ECC after sucrose rinse (p = 0.04), whereas mechanical biofilm control reduced it in both groups (p = 0.000). Phosphate concentration was influenced by mechanical control of biofilm in CF children (p = 0.03). The fluoride bioavailability was reduced by sucrose rinse and biofilm accumulation in CF and ECC children (p ≤ 0.002). 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.