Lactobacillus iners is often associated with increased risk of adverse gynecological and obstetric outcomes, such as vaginal dysbiosis and bacterial vaginosis (BV). Lactobacillus strains from the vagina of healthy women were screened for inhibitory activity against L. iners. Four active strains were identified, whereby L. gasseri H87 exhibited the highest inhibitory activity against L. iners, followed by L. rhamnosus H23. All four strains inhibited the growth of potential pathogens of the gastrointestinal, urogenital and reproductive tract, and were capable of co-aggregating with the cells of the tested pathogens to varying degrees. H23 and H78 exhibited survival rates above 80% under in vitro conditions simulating the vaginal, gastric and intestinal environment. The four strains showed good auto-aggregation of more than 30%. H23 and H87 also exhibited good co-aggregation of more than 30% with Listeria monocytogenes and Escherichia coli. Safety assessment showed that the four strains had the usual antibiotic susceptibility profile and did not produce hemolysins. Considering that H87 exhibited a better performance as a probiotic, we investigated the antibacterial substance produced by H87, and identified a bacteriocin with a molecular weight of 6.5 kDa. Based on strain characteristics and beneficial properties, we confirmed its ability to prevent/treat vaginal dysbiosis and maintain a healthy vaginal ecosystem. Therefore, we believe that H87 is a promising candidate for human trials.