Background: One of the causative factors in development of dental caries is microorganisms. Two species of Mutans streptococci including Streptococcus mutans and Streptococcus sobrinus are associated with dental caries in human beings. The aim of this study was to investigate the frequency of S. mutans and S. sobrinus in saliva of children with different caries activity and ability to form biofilm and acid susceptibility of these microorganisms. Materials and Methods: This analytical case-control study was performed on 83 preschool children, 4-6 years old. Children were divided into two groups including 41 caries-active and 42 caries-free children. Non-stimulated saliva samples were collected and culture and polymerase chain reaction techniques were used. Statistical analysis was performed using t-test, Chi-square, ANOVA, and Kappa tests. Results: S. mutans and S. sobrinus were found in 65% and 21.6% of the samples respectively. S. mutans was isolated from 75.6% of caries-active and 54.8% of caries-free children. Figures for S. sobrinus were 29.2% and 14.3% respectively. Acid susceptibility of microorganisms isolated from saliva was 87.43 in caries-active children and 94.30 for caries-free children. Biofilm formation of microorganisms in caries-active and caries-free children was 0.77 and 0.73, respectively. Conclusion: Frequency of S. mutans in caries-active children was significantly higher than caries-free children, but the difference in frequency of S. sobrinus was not significant. Acid susceptibility of microorganisms in caries-active children was significantly lower, but the ability to form biofilm was not significantly different in two groups.