Simple SummaryClinically,Streptococcus suisandHaemophilus parasuisoften co-occur or mix with each other, causing great harm to the pig industry. Thus, we established a mixed infection model in vitro and a co-infected mice model. We found that the co-existence ofS. suisandH. parasuiscan interfere with each other. There was competition betweenS. suisandH. parasuisin co-culture. Compared to single cultures, co-cultures showed enhanced biofilm formation, changes in virulence genes, and increased resistance to drugs. The number of bacteria in the co-infected mice increased and the inflammatory response changed. Ultimately, the study elucidated the interaction betweenS. suis and H. parasuis. This provides new ideas for the prevention and treatment of porcine respiratory disease syndrome caused by bacteria.AbstractStreptococcus suis(S. suis) andHaemophilus parasuis(H. parasuis) are two primary pathogens currently affecting the porcine industry. They often cause encephalitis and arthritis. They also frequently co-infect in clinical settings. In the current study, we identified significant correlations betweenS. suisandH. parasuis. The results from CI versus RIR suggested thatS. suisandH. parasuiswere competitive in general. Compared to mono-species biofilm, the biomass, bio-volume, and thickness of mixed-species biofilms were significantly higher, which was confirmed using crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. Compared to mono-species biofilm, the viable bacteria in the mixed-species biofilms were significantly lower, which was confirmed using the enumeration of colony-forming units (CFU cm−2). The susceptibility of antibiotics in the co-culture decreased in the planktonic state. In contrast, biofilm state bacteria are significantly more difficult to eradicate with antibiotics than in a planktonic state. Whether in planktonic or biofilm state, the expression of virulence genes ofS. suisandH. parasuisin mixed culture was very different from that in single culture. Subsequently, by establishing a mixed infection model in mice, we found that the colonization of the two pathogens in organs increased after mixed infection, and altered the host’s inflammatory response. In summary, our results indicate thatS. suisandH. parasuiscompete when co-cultured in vitro. Surprisingly,S. suisandH. parasuissynergistically increased colonization capacity after co-infection in vivo. This study elucidated the interaction betweenS. suisandH. parasuisduring single infections and co-infections. Future studies on bacterial disease control and antibiotic treatment should consider the interaction of mixed species.