Human vagina contains lactobacilli that are the part of vaginal microbiota. The identification and assessment of the unique properties of usual Lactobacillus species in the vagina of healthy women in various topographical locations will assist in selecting the best species. In the current study we have carried out the isolation and identification of Lactobacillus species from the vagina of healthy women to explore and evaluate their probiotic and antimicrobial efficacy. The research comprises of 30 women and bacteriawas isolated from the healthy individual were purified. The Lactobacillus was identified by the Phenotypic and biochemical tests. Lactobacillus species were identified by molecular method that is the amplification of the full length of the 16S rDNA of the isolated bacteria using polymerase chain reaction. In this study, eight vaginal lactic acid bacillus isolates manifested plethora of in vitro probiotic characteristics and showed potential activity against the most prevalent bacterial pathogens.