A biofilm is an aggregate of microorganisms in which cells adhere to biological or non-biological surfaces and is responsible for various infectious diseases. Infections caused by , including pneumonia, endocarditis, and osteomyelitis, are often associated with colonization and biofilm formation. Although lipoteichoic acid (LTA) is involved in biofilm formation, the specific role of LTA is not clearly understood. In this study, we demonstrated that LTA released from could inhibit biofilm formation and aggregation without affecting the growth of in various and models. LTA (Lp.LTA) also inhibited biofilm formation of clinical isolates, including a methicillin-resistant strain. Remarkably, Lp.LTA not only interfered with biofilm formation, but it also disrupted a pre-formed biofilm. Mechanism studies demonstrated that Lp.LTA inhibited expression of the -operon, which is responsible for the production of poly- -acetylglucosamine, a key molecule required for biofilm development. Lp.LTA increased the release of autoinducer-2 from , which contributed to the inhibition of biofilm formation. Moreover, Lp.LTA treatment enhanced susceptibility of the biofilm to various antibiotics and to macrophages. Interestingly, Lp.LTA without D-alanine moieties was not able to inhibit biofilm formation by . In conclusion, the present study suggests that LTA can inhibit biofilm formation, and therefore could be applied for preventing and/or treating infectious diseases caused by biofilms.