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Abstract: We report disposable microfluidic devices harboring an array of unsealed reactors, which are pre-loaded with different primer pairs for simultaneous (parallel) real-time PCR-based detection of multiple waterborne pathogens. The PCR mixture loading among an array of reactors and subsequent isolation of the reactors was solely realized by a single step capillary-based flow scheme, which eliminates the use of pumps, valves and liquid handling instruments. We incorporated a localized thermal cycling scheme to minimize evaporative loss of PCR mixture in unsealed microreactors, which greatly reduces the complexity of the microfabrication and fluidic operation process, in cases, where valving or sealing of the reactors for PCR thermal cycling is required. Experiments were performed to determine the optimal microreactor design parameters, so as to perform bubble-free PCR with minimal evaporative loss in unsealed reactors. The potential of the microfluidic device was successfully demonstrated by specifically detecting genomic DNA of waterborne pathogens from a pool of genomic DNA template. [Copyright &y& Elsevier]