Histophilus somni and Pasteurella multocida are two of multiple agents responsible for bovine respiratory disease (BRD) in cattle. Following respiratory infection of calves withH. somni,P. multocidamay also be isolated from the lower respiratory tract. BecauseH. somnimay form a biofilm during BRD, we sought to determine ifP. multocidacan co-exist withH. somniin a polymicrobial biofilmin vitroandin vivo. Interactions between the two species in the biofilm were characterized and quantified by fluorescencein situhybridization (FISH). The biofilm matrix of each species was examined using fluorescently tagged lectins (FTL) specific for the exopolysaccharide (EPS) using confocal laser scanning microscopy. Bacterial interactions were determined by auto-aggregation and biofilm morphology.Pasteurella multocidaandH. somniwere evenly distributed in thein vitrobiofilm, and both species contributed to the polymicrobial biofilm matrix. The average biomass and biofilm thickness, and the total carbohydrate and protein content of the biofilm, were greatest when both species were present. Polymicrobial bacterial suspensions auto-aggregated faster than single species suspensions, suggesting physical interactions between the two species. Almost 300P. multocidagenes were significantly differentially regulated when the bacteria were in a polymicrobial biofilm compared to a mono-species biofilm, as determined by RNA-sequencing. As expected, host genes associated with inflammation and immune response were significantly upregulated at the infection site followingH. somnichallenge. EncapsulatedP. multocidaisolates not capable of forming a substantial biofilm enhanced anin vitropolymicrobial biofilm withH. somni, indicating they contributed to the polymicrobial biofilm matrix. Indirect evidence indicated that encapsulatedP. multocidaalso contributed to a polymicrobial biofilmin vivo. Only the EPS ofH. somnicould be detected by FTL staining of bovine tissues following challenge withH. somni. However, both species were isolated and an immune response to the biofilm matrix of both species was greater than the response to planktonic cells, suggesting encapsulatedP. multocidamay take advantage of theH. somnibiofilm to persist in the host during chronic BRD. These results may have important implications for the management and prevention of BRD.