AbstractLactobacilli have been evaluated as probiotics against Candida infections in several clinical trials, but with variable results. Predicting and understanding the clinical efficacy of Lactobacillus strains is hampered by an overall lack of insights into their modes of action. In this study, we aimed to unravel molecular mechanisms underlying the inhibitory effects of lactobacilli on hyphal morphogenesis, which is a crucial step in C. albicans virulence. Based on a screening of different Lactobacillus strains, we found that the closely related taxa L. rhamnosus, L. casei and L. paracasei showed stronger activity against Candida hyphae formation compared to other Lactobacillus species tested. By exploring the activity of purified compounds and mutants of the model strain L. rhamnosus GG, the major peptidoglycan hydrolase Msp1, conserved in the three closely related taxa, was identified as a key effector molecule. We could show that this activity of Msp1 was due to its ability to break down chitin, the main polymer in the hyphal cell wall of C. albicans. This identification of a Lactobacillus-specific protein with chitinase activity having anti-hyphal activity will assist in better strain selection and improved application in future clinical trials for Lactobacillus-based Candida-management strategies.