Twenty lactobacilli isolated from vaginal samples of healthy volunteers were characterized according to polyphasic taxonomy. A broad spectrum of activity and protective properties of these vaginal isolates has been reported. Phenotypic and genotypic methods such as random amplified polymorphic DNA (RAPD), species-specific and BOX polymerase chain reaction (PCR), amplified ribosomal DNA restriction analysis (ARDRA), and 16S rRNA sequence analyses were applied for Lactobacillus spp. identification. On the basis of carbohydrate utilization profiles using API 50 CHL kits (fermentation pattern), the strains were divided into 7 groups. RAPD- and BOX-PCR analyses revealed heterogeneity within the established phenotypic groups and discriminated successfully between the vaginal strains. The combination of species-specific PCR, ARDRA, and 16S rRNA sequence analyses allowed the species identification of 7 strains as Lactobacillus fermentum, 2 as Lactobacillus gasseri, 1 as Lactobacillus brevis, and 1 as Lactobacillus salivarius. These results were not in agreement with data obtained by classical and molecular methods for some clinical isolates. This is the first polyphasic taxonomy study on vaginal lactobacilli from Bulgarian women of reproductive age. These data add to the limited information existing in Bulgaria on the true identity of Lactobacillus spp. in the vaginal microbiota.