In Egypt, turkey sinusitis is a major phenomenon that make the turkey farmers take far away from the turkey production as a result of difficult treatment, medication cost, difficult isolation of causative bacterial agents, weight loss, reduced fertility, and hatchability, so this study aimed to determine the bacterial causative agents using multiplex PCR as an accurate rapid diagnostic technique. Exudates of swollen infra-orbital sinus were aspirated and aseptically collected from affected turkeys of seventeen flocks and examined by multiplex PCR against Escherichia coli (E. coli), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma meleagridis (M. meleagradis), Ornithobacterium rhinotracheale (ORT), Pasteurella multocida (P. multocida), and Pseudomonas aeruginosa (P. aeruginosa) . The most prevalent detected bacteria were E coli (13) followed by M. gallisepticum (12), ORT (12) and M. meleagridis (7). Two farms reported single infection, one with M. gallisepticum and one with E. coli. Only one farm had no infection while mixed infection recorded in 14 farms. P. multocida and P. aeruginosa were not detected. It was concluded that Balady breed had a higher resistance than other breeds, E. coli and/or M. gallisepticum were the primary cause of turkey sinusitis, M. meleagridis recorded the lowest incidence and frequency in examined turkey flocks, while ORT had a higher incidence and frequency similar to M. gallisepticum. It was necessary to apply further molecular studies on these pathogens to control and avoid their spread in turkey and other poultry flocks.