Oral cancer is one of the leading causes of death worldwide, and its prevalence is especially high in developing countries. As an oral cancer treatment, traditional therapies are commonly used. Nonetheless, these treatments frequently result in a variety of side effects. As a consequence, there is an urgent need to enhance oral cancer therapies. Probiotics have recently demonstrated intriguing properties as therapeutic options for cancer treatment. Thus, the purpose of this study was to investigate the anticancer effect of probiotic Lactobacillus strains on the mouth epidermal carcinoma cells (KB) and oral squamous cell carcinoma (OSCC) cell lines. In this study, we looked at 21 Lactobacillus strains isolated from traditional dairy products in the Kermanshah province of western Iran to see if they had any inhibitory effects on oral cancer cell lines in vitro. We isolated and characterized Lactobacillus strains before assessing and comparing their probiotic potential and safety. Using the MTT assay, the bacterial extract was then prepared and used as an anti-proliferative agent on oral cancer (KB and OSCC) and normal (fibroblast and human umbilical vein endothelial cells (HUVEK) cell lines. Finally, acridine orange/ethidium bromide staining was used to determine whether cell death was caused by apoptosis. Four Lactobacillus isolates (C14, M22, M42, and Y8) were shown to have beneficial probiotic qualities. Lactobacillus extracts (of a protein nature) decreased the survival and proliferation of the KB and OSCC cancer cell lines (dose- and time-dependent) by inducing apoptosis, with no basic damaging effects on normal cells. The staining with acridine orange/ethidium bromide revealed that the cell death was caused by apoptosis. Furthermore, of the four Lactobacillus strains examined, isolate Y8 (Lactiplantibacillus plantarum) showed the strongest probiotic potential for suppressing KB and OSCC cell proliferation when compared to anticancer medicines (doxorubicin and paclitaxel). The current research found that Lactobacillus extract might reduce the growth and viability of the KB and OSCC cancer cell lines by inducing apoptosis, increasing the survival rate of oral cancer patients.