Aims:The aim was to complement the evaluation of functional and safety properties of beneficial vaginal lactic acid bacteria to select the most adequate strains as potential probiotics for their inclusion in a vaginal probiotic formula. Methods and Results:Twenty-four beneficial vaginal lactic acid bacteria strains previously isolated from healthy women were characterized by applying phenotypical and genetic techniques. The biofilm formation and exopolysaccharide production by the different strains showed a strong influence of the growth medium on the expression of these properties. The evaluated strains evidenced different antibiotic susceptibility patterns by phenotypic tests, while the resistance genes were not always correlated with the phenotypic profiles. None of the evaluated strains showed lecithinase, gelatinase, or βhemolytic activity. Based on the results obtained, Lactobacillus gasseri CRL 1320 and Lacticaseibacillus rhamnosus CRL 1332 were selected to advance in the design of the products. Conclusions:A wide variability in the properties expressed by beneficial vaginal lactic acid bacteria strains were shown, evidencing a strain-specific and independent behaviour of the species/metabolic group where classified. The selection criteria applied provided a valuable tool for the selection of the best vaginal lactobacilli strains for their inclusion in formulas for vaginal application. Significance and Impact of the Study: Since probiotics are an important tool for improving human health, the designed formula are an excellent alternative to reconstitute the vaginal microbiota and prevent/treat urogenital tract infections in women. The results obtained contribute to deepen the characterization of homologous beneficial vaginal lactic acid bacteria and advance in the design of vaginal probiotic products.