The vaginal microbial composition of wellbeing women were predominated by Lactobacillus group which play prime character in health-promoting activities including the prevention of the colonization of pathogenic microbe. The present study focused to characterize the probiotic and cytotoxicity potential of the Lactobacillus strains collected from the vaginal environment of the healthy women. Lactobacillus strains were identified via biochemical characterization and 16SrRNA gene sequencing. Strains were also evaluated for their probiotic potentials that includes antibiotics sensitivity; survivability to gastrointestinal conditions; antibacterial activities, biofilm production, auto aggregation and adhesion abilities to hydrocarbon. Cytotoxic activities were assessed through 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay using HeLa (cervical), AGS (gastric cancer) and A549 (lung cancer) cell lines. Results identified five bacterial strains viz., (Lactobacillus mucosae K76, L. fermentum K81, L. fermentum K85, L. reuteri K97 and L. reuteri K99) contains high probiotic potential and three strains with high cytotoxic activities against cancer cell lines can be further explodes for the human beneficial activities. Finding from this study also indicates that vaginal environment represents a novel source of probiotic candidates.