Bacterial pathogens play an important role in causing respiratory diseases in poultry. Six pathogens; Escherichia coli, Mycoplasma gallisepticum, Staphylococcus aureus, Pasteurella multocida, Mannheimia haemolytica and Pseudomonas aeruginosa are known to cause respiratory infections in poultry. However, current diagnostic methods for them are not highly sensitive, laborious and time consuming. This research aimed to develop a multiplex polymerase chain reaction (m-PCR) that could detect the six species in clinical specimens from poultry with respiratory manifestations. In particular,  E. coli alkaline phosphatase (phoA), S. aureus encoding a surface associated fibrinogen binding protein (clfA), P. aeruginosa outer membrane protein (oprL), M. haemolytica serotype specific antigen (SSA), M. gallisepticum 16S rRNA and P. multocida ribosomal RNA(23S rRNA) genes were tested. PCR Results revealed that 19 (35.8%) out of 53 examined farms were with a single infection, 31 (58.5%) associated with mixed bacterial infections, meanwhile 3 only (5.7%) were negative. The m-PCR developed in this study has been proven to be both sensitive and specific for simultaneous detectionof E. coli, M. gallisepticum, S. aureus and P. aeruginosa in clinical specimens in a single reaction. However, detection of M. haemolytica was suboptimal due to indistinguishable fragment size and lower annealing temperature. Overall, standardized m-PCR in our research could be considered as a useful tool for diagnosis and screening of E. coli, M. gallisepticum, S. aureus and P. aeruginosa in poultry flocks as it takes almost three hours to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process.