Nosocomial infections are a major public health concern worldwide. Early and accurate identification of nosocomial pathogens which are often multidrug resistant is crucial for prompt treatment. Hence, an alternative real-time polymerase chain reaction coupled with high resolution melting-curve analysis (HRMA) was developed for identification of five nosocomial bacteria. This assay targets species-specific regions of each nosocomial bacteria and produced five distinct melt curves with each representing a particular bacterial species. The melting curves were characterized by peaks of 78.8 ± 0.2 °C for Acinetobacter baumannii, 82.7 ± 0.2 °C for Escherichia coli, 86.3 ± 0.3 °C for Klebsiella pneumoniae, 88.8 ± 0.2 °C for Pseudomonas aeruginosa and 74.6 ± 02 °C for methicillin-resistant Staphylococcus aureus. The assay was able to specifically detect the five bacterial species with an overall detection limit of 2 × 10− 2 ng/μL. In conclusion, the HRM assay developed is a simple and rapid method for identification of the selected nosocomial pathogens.