Despite recent advances in the miniaturization and automation of biosensors, technologies for on-site monitoring of environmental water are still at an early stage of development. Prevention of outbreaks caused by pathogens such as Legionella pneumophila would be facilitated by the development of sensitive and specific bioanalytical assays that can be easily integrated in miniaturized fluidic handling systems. In this work, we report on the integration of an amplification-free assay in digital microfluidics (DMF) for the detection of Legionella bacteria based on targeting 16s rRNA. We first review the design of the developed DMF devices, which provide the capability to store up to one hundred nL-size droplets simultaneously, and discuss the challenges involved with on-chip integration of the RNA-based assay. By optimizing the various steps of the assay, including magnetic capture, hybridization duration, washing steps, and assay temperature, a limit of detection as low as 1.8 attomoles of synthetic 16s rRNA was obtained, which compares advantageously to other amplification-free detection systems. Finally, we demonstrate the specificity of the developed assay by performing multiplex detection of 16s rRNAs from a pathogenic and a non-pathogenic species of Legionella. We believe the developed DMF devices combined with the proposed detection system offers new prospects for the deployment of rapid and cost-effective technologies for on-site monitoring of pathogenic bacteria.