Fish with bacterial septicemic syndrome (BSS) exhibit very similar clinical signs regardless of the etiological agents. Members of Aeromonas, Pseudomonas, Vibrio, Edwardsiella, Streptococcus and Lactococcus species are considered the most reported bacterial pathogens incriminated in such syndrome. Aeromonas hydrophila, Edwardsiella tarda and Streptococcus iniae are 3 major pathogens share in the BSS associated losses in aquaculture and considered problematic for growth of tilapia and catfish production in Egypt. Therefore, rapid and accurate diagnosis is highly needed for controlling their disease outbreaks, particularly, in mixed infections. In an attempt to elucidate the main causative pathogen, a novel multiplex PCR (m-PCR) was newly designed in this study. The developed m-PCR involves amplifying the three multiple genes in single reaction based upon primers deduced from the regions carrying 16S rRNA, etfA and 16S RNA genes of A. hydrophila (Aeromonas spp.), E. tarda and S. iniae, respectively. Prior to perform m-PCR, individual PCR assays were carried out to adapt suitable laboratory and m-PCR assays conditions. The specificity of the developed m-PCR was confirmed by the fact that only specific fragments were amplified equivalent for 953, 415 and 300 bp corresponding to A.hydrophila, E. tarda and S. iniae, respectively, and that was evident with both extracted DNAs and the bacterial cells. More specifically, these specific bands were obtained also when either the extracted DNAs or the bacterial cells of the three pathogens mixed together in the reaction. The developed m-PCR is accurate, sensitive, fast and simple technique for the simultaneous detection of A. hydrophila (Aeromonas spp.), E.tarda, and S. iniae, three major bacterial pathogens involved in BSS incidence in Egypt.