To fully utilize EBC in early disease screening, diagnosis and environmental exposure assessment, simple yet efficient EBC collection device using different methods and biological characterization of the EBC sample are needed. In this study, a novel EBC collection method was developed by using hydrophobic surface, a layer of ice, and a droplet scavenging procedure. The physical collection efficiency of the device was evaluated. In addition, biological analysis and characterization of EBC samples collected from human subjects were conducted using culturing, DNA stain, SEM, qPCR and species identification tool VITEK 2. This work contributes to the effort in applying EBC together with molecular tools as a non-invasive method in rapid disease diagnosis. For negative control samples, we did not observe the bacterial growth, indicating no contamination during the EBC collection. Ideally, bacterial particles in EBC should be collected using a suitable size-selective sampling tool to investigate the bacterial counts for different size range. Torin 1 mTOR inhibitor However, such device is currently not available yet. Compared to the environmental culturable bioaerosol concentrations, those in EBC samples collected had relatively higher levels, thus representing an important source of bioaerosols particularly in a high human occupancy environment. In addition to viruses, Rhodococcus equi, a bacterium causing pyogranulomatous bronchopneumonia, were detected in the exhaled air from foals in a recent study. When pathogenic bacteria are breathed out, they could pose a serious public health threat. Exhaled breath holds great promise for monitoring human health and for the diagnosis of various lung and systemic diseases, but analysis challenges remain due to the complex matrix of the breath. In this study, different from available devices restricted solely to condensation a simple and low cost EBC collection method using impaction and condensing was developed here for collecting bacteria and virus particles. An important advantage is the reusability of the collection device with a disposable hydrophobic film and an exhalation straw yet with a rapid EBC collection. This would offer the opportunity to collect EBC samples from a large number of subjects, especially during an influenza outbreak or a man-made bioterrorism event, within a shorter time frame. The developed EBC collection method was shown highly successful in detecting bacteria in EBC samples in a clinical setting. The developed EBC collection method was also shown applicable in detecting influenza viruses too. Experimental data here also suggest that exhaled breath, which was shown to contain smaller bacterial particles, could play an important role in airborne transmission of potential diseases. The collection efficiency of other substances including bio-markers using the developed method here is subject to further investigations. In addition, different exhalation modes should be also investigated with the method in collecting EBC. Besides, the dynamics of the air flow, mixing, and effects of temperatures and humidity, condensation, evaporation, growth of particles during the collection as well as the optimal straw length should be also investigated for improving the developed technique. Overall, our developed method here could be easily made available to a laboratory, and have impacts on current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of those influencing factors described.