ABSTRACTBiofilms contribute toPseudomonas aeruginosapersistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressedP. aeruginosabiofilmsin vivo. We used a chinchilla model of otitis media, which has previously been used to study persistentStreptococcus pneumoniaeandHaemophilus influenzaeinfections, to show that structures formedin vivoare biofilms of bacterial and host origin within a matrix that includes Psl, aP. aeruginosabiofilm polysaccharide. We evaluated three biofilm and/or virulence mediators ofP. aeruginosaknown to affect biofilm formationin vitroand pathogenesisin vivo—bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing—in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators ofP. aeruginosaand thatin vitrophenotypes should be examined in multiple infection systems to fully understand their role in disease.