In the present study, twenty-seven (27) lactobacilli strains, isolated from the vagina of healthy Italian women of reproductive age, were screened for probiotic properties. The strains were evaluated for antagonistic activity against pathogens, adhesion abilities, and potential to displace and/or inhibit the adhesion of previously adhered pathogens as a primary strain selection criterion. Overall, all the tested lactobacilli inhibited at least three pathogens, and the majority of them exhibited antimicrobial activity against Enterobacter cloacae DSM 30054, Pseudomonas aeruginosa DSM 3227, and Pseudomonas aeruginosa DSM 1117. The complete neutralization of antimicrobial activity after cell-free supernatant (CFS) neutralization suggested a pivotal role for lactic acid or other organic acids secreted by the lactobacilli. The strains showed variability in their adhesion levels, but all tested strains adhered to both human colonic epithelial cells (HT-29) and vaginal cells (VK2/E6E7) with adhesion percentages exceeding 1%. The ability to displace or inhibit pathogens was dependent on the pathogen and the lactobacilli strain; the pathogen displacement levels ranged from 9 to 82%, while pathogen exclusion levels varied from 1 to 99%. In conclusion, this study demonstrates the protective effect of vaginal lactobacilli against pathogens and confirms the suitability of the vaginal microbiota as a source of potential probiotic strains. The selected lactobacilli hold promise for the formulation of supplements to enhance genitourinary tract health.