Aims The aim of this study was to characterize Lactobacillus strains for their biological properties and amensalistic activities against genital and nongenital pathogens. Methods and Results For the purpose, some special characteristics (H2O2, biofilm and antimicrobial substances production) as well as safety properties of 112 lactobacilli were evaluated. All the strains had good amensalistic characteristics, in particular cell-free supernatants of 10 strains showed antibacterial activity against bacteria, as well as Candida sp. Moreover, these 10 strains were excellent biofilm producers. Conclusions These results provide evidence for the possible use as probiotics for vaginal co-therapy in case of dysbiosis. Significance and Impact of the Study Recently, the problem of antibiotic resistance is constantly increasing, even though resources and energy are invested in order to increase knowledge on the mechanisms of action. Bacteriocins have a rapid mechanism of action, act at extremely low concentrations, are generally sensitive to proteases and they usually have a narrow killing spectrum; these characteristics reduce the possibility of the bacterium to develop resistance. This study is focused on the feasibility of a high production of antimicrobial substances and their characterization in order to be exploited as a therapeutic alterative or in co-therapy with antibiotics in case of vaginal dysbiosis.