Oral illnesses such as dental caries and gingivitis are frequent. In these kinds of infection, the bacteria are thought to use biofilm formation as a pathogenic mechanism. Today, due to the side effects of chemical medications and resistance to antibiotics, the use of probiotics is of great importance as a suitable alternative way for infection treatment. Probiotics have shown favourable properties in terms of maintaining oral health. Kefir, which is made up of complex microbiota mainly Lactobacillus spp., has been suggested as a potential reservoir for probiotic. The present research aimed to isolate and identify lactic acid bacteria from traditional kefir dough as a probiotic that suppresses S. mutans and P. gingivalis growth, biofilm formation, and gene expression. In this study, Lactobacillus spp. was tested for antibacterial (well agar diffusion technique) and antibiofilm (crystal violet assay) properties against Streptococcus mutans (ATCC 35668) and Porphyromonas gingivalis (ATCC 33277). Through the use of real-time polymerase chain reaction, we explored whether Lactobacillus plantarum and Lactobacillus rhamnosus inhibited expression of Streptococcus mutans genes involved in biofilm formation and stress survival. Additionally, Lactobacillus plantarum and Lactobacillus rhamnosus dramatically inhibited the expression of gtfB and brpA.