Lactobacillus species are the predominant vaginal microbiota found in healthy women of reproductive age and help to prevent pathogen infection by producing lactic acid, H2O2 and anti-microbial compounds. Identification of novel vaginal Lactobacillus isolates that exhibit efficient colonisation and secrete anti-Candida factors is a promising strategy to prevent vulvovaginal candidiasis. The azole antifungal agents used to treat vulvovaginal candidiasis elicit adverse effects such as allergic responses and exhibit drug interactions. Candida strains with resistance to antifungal treatments are often reported. In this study, we isolated Lactobacillus species from healthy Korean women and investigated their antifungal effects against C. albicans in vitro and in vivo. Lactobacillus conditioned supernatant (LCS) of L. crispatus and L. fermentum inhibited C. albicans growth in vitro. A Lactobacillus-derived compound, which was not affected by proteolytic enzyme digestion and heat inactivation, inhibited growth and hyphal induction of C. albicans after adjustment to neutral pH. Combination treatment with neutral LCSs of L. crispatus and L. fermentum effectively inhibited propagation of C. albicans in a murine in vivo model of vulvovaginal candidiasis.