PE-PilA is a fusion protein composed of immunologically relevant parts of protein E (PE) and the majority subunit of the type IV pilus (PilA), two major antigens of nontypeable Haemophilus influenzae (NTHi). Here we report on the preclinical evaluation of PE-PilA as a vaccine antigen. The immunogenic potential of the PE and PilA within the fusion was compared with that of isolated PE and PilA antigens. When injected intramuscularly into mice, the immunogenicity of PE within the fusion was equivalent to that of isolated PE, except when it was formulated with alum. In contrast, in our murine models PilA was consistently found to be more immunogenic as a subentity of the PE-PilA fusion protein than when it was injected as an isolated antigen. Following immunization with PE-PilA, anti-PE antibodies demonstrated the same capacity to inhibit the binding of PE to vitronectin as those induced after PE immunization. Likewise, PE-PilA-induced anti-PilA antibodies inhibited the formation of NTHi biofilms and disrupted established biofilms in vitro These experiments support the immunogenic equivalence between fused PE-PilA and isolated PE and PilA. Further, the potential of PE-PilA immunization against NTHi-induced disease was evaluated. After intranasal NTHi challenge, colonization of the murine nasopharynx significantly dropped in animals formerly immunized with PE-PilA, and in chinchillas, signs of otitis media were significantly reduced in animals that had received anti-PE-PilA antibodies. Taken together, our data support the use of PE-PilA as an NTHi vaccine antigen.