The vaginal microbiota of healthy women is dominated by lactobacilli, which exerts important health-promoting effects to the host. In the present study, 261 lactobacilli isolated from vagina of healthy women were screened for their potential probiotic characteristics. Safety features (haemolytic activity, antibiotic susceptibility, bile salt hydrolase activity) and functional properties (resistance to low pH and bile salts, lysozyme tolerance, gastrointestinal survival, antagonistic activity against pathogens, hydrophobicity, auto-aggregation, and co-aggregation abilities, hydrogen peroxide production, biofilm formation, exopolysaccharide production, adhesion capacity to both normal human vagina epithelial cells and Caco-2 epithelial cells, and lactic acid production) were in depth evaluated. Seven strains, identified as Lactobacillus rhamnosus, Lactobacillus helveticus and Lactobacillus salivarius fulfilled the criteria described above. Therefore, the vaginal ecosystem represents a suitable source of probiotic candidates that could be used in new functional formulates for both gastrointestinal and vaginal eubiosis.