The usage of probiotics is becoming more common because of the mounting body of research demonstrating its advantages for human health. Of late probiotics have transitioned to a highly promising prophylactic measure that affords colonization resistance against multitude of pathogens. A crucial defence mechanism in the human body against numerous illnesses, especially urogenital ones, is the vaginal microbiota. The present study focuses on screening and isolation of culturable vaginal commensal microbiota from 54 healthy Indian women, and explore its putative probiotic traits such as coaggregation, antagonistic action against uropathogens, ability to form biofilms, antibiotic sensitivity, adhesion properties, etc. In addition, how oestradiol might affect isolated vaginal probiotics in both planktonic and biofilm states were also explored. The purpose of this work was to build a preliminary probiotics topography, specifically a Lactobacilli cartography, from a healthy Indian women cohort, characterize them at the molecular level, and interpret the key activities which were needed for the design and use probiotics as an antimicrobial sparing therapy to curtail UTI infection on the face of rising antimicrobial resistance. Our long-term goal of this work is to develop feminine hygiene product that is incorporated with probiotics to protect against pathogen colonisation without creating dysbiosis and improve women's quality of life.