Otitis media (OM) is among the most common childhood diseases worldwide. OM infections often persist for long periods of time and can be highly resistant to antibiotic treatment due in part to persistence of bacteria within biofilms. The majority of OM infections also involve coinfection by multiple species. For instance, the leading causative agents Haemophilus influenzae and Moraxella catarrhalis are more frequently found to coexist than to be isolated individually. Polymicrobial infection and biofilm formation may have a dramatic impact on the severity and treatability of OM. Thus, there is a pressing need to investigate polymicrobial infection and to identify factors that contribute to the establishment of these infections. One factor that may contribute to the establishment of polymicrobial infection is bacterial communication via quorum signaling. Autoinducer-2 (AI-2) is termed an interspecies signal as the genetic determinant for AI-2 production (luxS) is highly conserved and many species possess the machinery to respond to AI-2. H. influenzae has a luxS homolog whereas M. catarrhalis does not but may still sense and respond to AI-2. Therefore, the focus of this project was to investigate the impact of AI-2 intra- and interspecies bacterial communication and H. influenzae and M. catarrhalis polymicrobial infection on biofilm formation, antibiotic resistance, and persistence in experimental OM.