PURPOSE: The aim of this study was to identify novel vaginal probiotics with the potential to prevent vulvovaginal candidiasis (VVC) and bacterial vaginosis (BV). MATERIALS AND METHODS: Eighteen strains of Lactiplantibacillus plantarum were isolated from healthy Korean women, and their antimicrobial effects against Candida albicans and Gardnerella vaginalis were assessed. Three strains (L. plantarum LM1203, LM1209, and LM1215) were selected for further investigation, focusing on their growth inhibition, biofilm regulation, and cellular mechanisms against these vaginal pathogens. Additionally, electron microscopy revealed damage to G. vaginalis induced by L. plantarum LM1215, and genomic analysis was conducted on this strain. RESULTS: L. plantarum LM1203, LM1209, and LM1215 showed approximately 1 and 2 Log CFU/mL growth reduction in C. albicans and G. vaginalis, respectively. These L. plantarum strains effectively inhibited biofilm formation and eliminated the mature biofilms formed by C. albicans. Furthermore, L. plantarum LM1215 decreased tricarboxylic acid cycle activity by 51.75 (p<0.001) and respiratory metabolic activity by 52.88% (p<0.001) in G. vaginalis. L. plantarum induced cellular membrane damage, inhibition of protein synthesis, and cell wall collapse in G. vaginalis. Genomic analysis confirmed L. plantarum LM1215 as a safe strain for vaginal probiotics. CONCLUSION: The L. plantarum LM1215 is considered a safe probiotic agent suitable for the prevention of VVC and BV.