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The modern and rapid avenue for detecting pathogens provided by molecular genetic techniques including polymerase chain reaction (PCR) was explored in the present study to identify prevalent disease pathogens, from six aquaculture farms and in two commonly cultured fish in Ghana. The specific detection was carried out directly on clinical samples of naturally infected fish (O. niloticus and C. gariepinus) based on syber-mix reaction protocol in traditional PCR. Molecular diagnostic techniques allowed the detection of six most common and important bacteria pathogens in aquaculture farms in Ghana. Also, three of the pathogens (Streptococcus agalactiae, Streptococcus iniae and Staphylococcus aureus) were simultaneously isolated in a multiplex reaction. The results indicated 90% - 100% sensitivity and specificity for each of the six bacterial pathogens tested. Streptococcosis and motile aeromonad septicemia were found to be highly prevalent in most aquaculture farms in Ghana with severity in infections traced to the 85.7% and 14.9% co-infections with all six target pathogens in catfish and tilapia respectively. Prevalence rate of infections significantly correlated with variations in salinity, conductivity and dissolved oxygen concentrations in the thermal stressed condition of the culture water.