Background and Aims: Infectious diseases are affecting people more often than before and that is the main cause of morbidity and mortality in developed and developing countries. Taking the necessary measures by diagnosing earlier and using appropriate antibiotics made significant contributions to the control of the infections. In our study we aimed to develop a method for the diagnosis of some of pathogen gram-negative bacteria by using multiplex PCR (Polymerase Chain Reaction) from a positive blood culture bottles. Methods: Extraction method was applied from the blood cultures in which growth was detected. After mono¬plex PCR experiments were performed and images were obtained, multiplex PCR experiments were performed. Considering the product sizes and primers for multiplex PCR, a quadruple bacterial set was prepared by matching Acinetobacter baumannii, Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa. Experiment conditions and tube concentration were adjusted by making serial trial experiments. Results: Multiplex-PCR-based methods have the advantage of defining multiple pathogens in a single PCR reaction. These are used for identifying respiratory infections, bacteraemia, and meningitis which are common bacterial and viral diseases. Type-specific genes and molecular techniques including identification of infectious diseases with nucleic acid amplification are promising tools for the verification of accurate diagnosis. The feasibility of this assay in routine diagnostic testing was evaluated using 146 positive blood cultures and 40 negative blood culture samples. Conclusions: Early diagnosis and intervention with effective antimicrobial therapy is important in reducing mortality that is associated with bloodstream infections.