The aim of this study is to test the production of bacteriocin in vaginal Lactobacilli and determining the characterization and antibacterial activity of this bacteriocin. For this reason 100 Lactobacillus strains were isolated and identified from vaginal swab samples of 75 women who attended obstetric and gynecology clinics. It was determined that six of 100 Lactobacillus strains produced bacteriocin. The antibacterial activity of bacteriocins was examined and found to be effective on vaginal L. gasseri, L. acidophilus, Gardnerella vaginalis ATCC 14018 and Pseudomonas aeroginosa ATCC 10145. We also found that bacteriocins were sensitive to proteinase enzymes and alkalinity, but resistant to catalase. Two of them, TL059a and TL080, were resistant to chloroform and 1 h boiling. It was observed that adding 1% NaCl to medians increased bacteriocin production and it was also found that mitomycin C induced Rogosa SL media was more suitable than MRS (Z = -2, p < 0.05).