OBJECTIVE To quantify and compare Streptococcus mutans (S. mutans) and Lactobacillus fermentum (L. fermentum) in saliva and biofilm of caries-free children to those with cavitated and non-cavitated lesions. METHODS One hundred and thirty-five 3-4 years old children were grouped (n = 45 in each group) according to their caries status: Clinical examination was done by a calibrated examiner. Biofilm and saliva were collected to quantify the microorganisms using qRT-PCR. The decayed-missing-filled surfaces (dmfs) was calculated by adding the number of decayed (ICDAS-II score 3-6), filled (ICDAS-II score 7 and 8) and missing (ICDAS-II score 9) surfaces due to caries. The correlation between the bacterial amounts and the number of carious surfaces was evaluated using Spearman's correlation coefficient. The levels and proportions of the microorganisms were compared using the Kruskal-Wallis test at an α-level of 0.05. RESULTS The quantity of S. mutans and L. fermentum was significantly higher in saliva and biofilm of children with cavitated lesions, followed by those with non-cavitated lesions and the lowest in caries-free children. Also, salivary and biofilm S. mutans, along with biofilm L. fermentum levels, significantly correlated with the number of non-cavitated surfaces; while salivary and biofilm S. mutans and L. fermentum levels significantly correlated with the number of cavitated surfaces. Additionally, dmfs scores significantly correlated with the salivary and biofilm S. mutans and L. fermentum levels. CONCLUSIONS S. mutans and L. fermentum in saliva and biofilm samples are associated with caries lesion severity.