Avian arthritis is a relatively common disease in the poultry industry, the cause of which is complex. Bacterial arthritis is often caused by infection of Staphylococcus aureus(S. aureus), whereas viral arthritis is caused by avian orthoreovirus (ARV). To investigate the infection of S. aureusand ARV in cases of avian arthritis, a total of 77 samples characterized by arthritis were collected and detection. The results showed that 68.83% of the samples were positive for ARV, and 66.23% were positive for S. aureus. Among them, the ARV mono-infection rate was 22.08%, the S. aureusmono-infection rate was 19.48%, and ARV and S. aureusco-infection rate was 45.45%, indicating that ARV and S. aureusco-infection is common in arthritis cases. To further investigate the synergistic pathogenicity of ARV and S. aureus, ARV and S. aureuswere used to mono-infect, co-infect, and (or) sequential infect SPF chickens and the clinical indications, pathologic changes, ARV load, S. aureusbacterial distribution, and cytokine level of the challenged chickens were evaluated. Decreased weight gain, increased mortality, and difficulties in standing were observed in all dual-infected groups and the singular-infected group. There were significantly more severe macroscopic and microscopic hock lesions, and larger amounts of a wider range of tissue distribution of ARV antigens and S. aureusbacterial distribution in the dual-infected groups compared to the single-infected and control groups. Cytokine detection showed a significant change in IFN-γ, IL-1β, and IL-6 levels in the infected groups, especially in the ARV–S. aureusco-infection, and (or) sequential infection groups, compared with the control group. Hence, ARV and S. aureussynergistically increased mortality in infected chickens, potentiated the severity of arthritis, and increased the amount of ARV RNA in tendons.