A robust, real-time polymerase chain reaction (RT-PCR) system to universally detect microbes at a limit of 10 to 50 colony-forming units within 5-6 h was developed. Pre-treatment of RT-PCR master mixes with ethidiumbromide monoacide (EMA) facilitates the development of an RT-PCR assay with appropriate sensitivity, reproducibility, and recovery.The system is useful to replace conventional microbial plating techniques for the analysis of microbial contamination in liquids like water. This was statistically confirmed for eight different bacteria and two different fungi species. Finally a complete procedure including microbial lysis, DNA extraction, EMA treatment, and RT-PCR was developed and evaluated for three different bacteria and two fungi species.