Pasteurella multocida causes respiratory and multi-systemic diseases in multiple animal species. The glycosaminoglycan capsule of P. multocida protects the bacterium from host defenses. However, chronic P. multocida infections associated with bovine respiratory disease (BRD) and avian cholera may be associated with a biofilm. Our aims were to characterize biofilm formation by P. multocida, if biofilm contributes to chronic infections, and if P. multocida interacts with Histophilus somni in a poly-microbial biofilm. There was an inverse correlation between P. multocida biofilm formation and capsule production (determined by uronic acid and Congo Red uptake assays), which was confirmed with capsule-deficient mutants and isolates. The exopolysaccharide (EPS) matrix of the biofilm was determined to be glycogen by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzyme digestion. Encapsulated, biofilm-deficient strains caused acute avian cholera, whereas capsule-deficient, biofilm-proficient strains were associated with chronic infections. Histopathological exam showed that biofilm forming isolates induced little inflammation in the lungs, heart, and liver. Putative biofilm material was identified in pulmonary tissues of chickens with chronic avian cholera using fluorescence-tagged lectin (FTL) specific for the biofilm EPS. Quantitative real-time PCR for expression of cytokine genes in infected chicken spleens indicated that P. multocida induced Th1 and Th17 immune responses during acute and chronic avian cholera. H. somni and P. multocida formed a poly-microbial biofilm in vitro and in the bovine respiratory tract, determined by fluorescence in situ hybridization, FTL, and confocal scanning laser microscopy with COMSTAT z-stack image analysis. Encapsulated P. multocida isolates not capable of forming a biofilm still formed a poly-microbial biofilm with H. somni. Therefore, P. multocida was capable of forming a proficient biofilm if the isolates were capsule-deficient, and were more likely to be associated with chronic, less inflammatory infections. These results may have important implications for the management of infections due to P. multocida.