Background: This study aimed to evaluate the possible role of EBV/HPV co-infection as an etiologic factor in prostate cancer (PCa) development.Methods: The present case‐control research was conducted on 67 cases with prostate cancer and 40 controls. The expression of cellular and viral factors involved in inflammation, tumor progression, and metastasis were quantitated using ELISA and qRT-PCR.Results: The EBV/HPV co-infection was reported in 14.9% of case group and 7.5% of control group. The high-risk type of HPV, including HPV 16 and 18, were responsible for 50% and 30% of 10 HPV/EBV co-infected PCa samples, respectively. According to the results, a significant relationship was not observed between the PCa and HPV/EBV co-infection (OR=2.9, 95%CI=0.18-45.2, P=0.31). However, the highest percentage of HPV genome integration was found in HPV/EBV coinfected PCa group (8/10, 80%). Moreover, the mean expression levels of inflammatory factors (IL-17, IL-6, TNF-α, NF-κB, VEGF, ROS and RNS), anti-apoptotic mediators (Bcl-2 and Survivin), and anti-anoikis factors (TWIST, N-cad) were higher significantly in the HPV/EBV co-infected PCa cases when comparing with the non-coinfected PCa samples. Nevertheless, the tumor suppressor proteins (p53 and Rb) and the E-cad (inhibiting anoikis resistance) had a significant downregulation in the HPV/EBV co-infected PCa cases than in the non-coinfected PCa samples. Conclusion: HPV/EBV co-infection probably can act as an etiologic factor in PCa through modulation of cellular behavior.