Background: We evaluate the composition of vaginal lactobacilli in women with bacterial vaginosis (BV) before and after treatment with a nifuratel nystatin complex. Methods: This multicenter prospective observational study included 40 women with abnormal vaginal discharge and a confirmed diagnosis of BV based up on Amsel criteria who received treatment with vaginal capsules containing 500 mg Nifuratel and 200,000 IU nystatin. Quantitative assessment, with typing of Lactobacillus species using real-time PCR and vaginal pH were evaluated before and after treatment. The control group consisted of 25 women without vaginal discharge. Results: All women had a lactobacillus index (LI) which indicated the proportion of lactobacilli in the total bacterial mass, ranging from 0.01% to 100%, and had a significant diversity in lactobacillus species in both groups. The controls predominantly showed L. crispatus (56.0%), most samples contained two or three species of Lactobacillus and 80% of them had a high LI (>70%). Women with BV demonstrated dominance by L. iners (62.5%) and 32.5% of samples had a low LI (<30%). After the treatment, no women complained of vaginal discharge, and the LI gradually increased. Within 4-6 weeks, detection rates of L. iners, L. crispatus, L. jensenii and L. gasseri reached characteristics comparable to the control group. The vaginal pH was not an indicator of the cure of BV but correlated with the changes of composition of Lactobacilli. Conclusions: The nifuratel-nystatin complex demonstrated high efficacy in the treatment of BV and has contributed to the restoration of the eubiotic vaginal species of lactobacillus.