ABSTRACT Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5–6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosaand Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5–6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of nonsterile pharmaceutical products can be performed in a cost‐effective and timely manner in pharmaceutical industry.PRACTICAL APPLICATIONSDetection of pathogenic indicatiors of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of nonsterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost‐effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.