Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, Gram-negative bacteria, are considered major pathogens of AOM and respiratory tract infections. In this study, we used monophosphoryl lipid A (MPL) as a Toll-like receptor (TLR4) agonist to induce innate immune responses before challenge with NTHi and M. catarrhalis to enhance bacterial clearance from the nasopharynx. Mice were intranasally administered 40, 10, or 1 μg of MPL and challenged with NTHi and M. catarrhalis 12 and 24 h later. At 6 and 12 h after the bacterial challenge, the mice were killed and nasal washes were collected. The numbers of NTHi, M. catarrhalis, and inflammatory cells were quantitated. Inoculation of MPL produced a significant reduction in the number of bacteria recovered from the nasopharynx at 6 and/or 12 h after the bacterial challenge, when compared with control mice. The effect was dose dependent. MPL inoculation also induced the early accumulation of neutrophils in the nasopharynx after exposure to bacteria. MPL is effective for eliciting clearance of both NTHi and M. catarrhalis from the nasopharynx. These results indicate the possibility of a new strategy against Gram-negative bacterial infection that involves the stimulation of the innate immune system by TLR4 agonists such as MPL.