Streptococcus mutans, in relation to the expression of genes encoding the enzymes glucosyltransferases, leading to formation of biofilm has been associated with tooth decay. Several reports have suggested that species of Lactobacillus provide probiotic effect against oral cavity. The current research was therefore conducted to investigate the effect of Lactobacillus species on the expression of gene associated with biofilm formation by S. mutans. Eighty-six saliva swab samples were obtained from patients suffering from tooth decay. Streptococcus species was isolated from saliva samples and characterized using cultural and biochemical methods. Species identity of Streptococcus and Lactobacillus was confirmed by sequencing of the 16S rRNA gene. Biofilm formation assay was set up using culture of S. mutans (control) or co-cultures with either of the two species of Lactobacillus, respectively. RT-qPCR was used to examine the expressions of gtfB gene in all the incubated cell cultures. The results revealed that out of the 86 test saliva samples, 34.8% was positive for S. mutans. The molecular identification revealed the identities of the species of Lactobacillus to be L. salivarius and L. acidophilus. It was observed that both L. acidophilus and L. salivarius lowered biofilm formation by S. mutans to a significant level. Furthermore, there was an increase in the level of gtfB gene expression in S. mutans when co-cultured with L. salivarius (2.9-fold) or L. acidophilus (1.9-fold), compared with a lone culture of S. mutans. Our finding indicates the role of Lactobacillus species in the control of dental caries.