The significance of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in the poultry industry underscores the critical importance of diagnosing avian mycoplasmosis within the field of veterinary medicine. The present study aimed to compare various diagnostic methods for detecting MG and MS in Egyptian Chicken Flocks. A total of 360 samples were collected from breeder, layer, and broiler chickens from four governorates in Egypt. Conventional isolation methods and polymerase chain reaction (PCR) were used for the direct detection of MG and MS, while serum plate agglutination test (SPA) and Enzyme-linked immunosorbent assay (ELISA) were used for detecting antibodies against MG and MS. The highest detection rate of MG was found in commercial layers, followed by breeders, and broilers. Regarding MS, the highest detection rate was found in breeders, followed by commercial layers, and broilers. By comparing the used diagnostic methods, MG and MS were determined by the SPA test (40% and 31.1% respectively), ELISA test (31.7% and 23.6%), PCR (16.7% and 11.7%), and by the conventional culture method (10.8% and 3.9%). It could be concluded that the serological methods and PCR gave better sensitivity than culture methods and can be used in the diagnosis of avian mycoplasmosis.