Given the current problems associated with the COVID-19 pandemic, research in the field of rapid and reliable detection and characterization of airborne pathogenic microorganisms is becoming of exceptional urgency. This project is focused on development of portable first alert bioaerosol monitoring technique utilizing personal bioaerosol sampler capable of high efficient trapping of airborne microorganisms into collecting liquid along with a laser based mini Polymerase Chain Reaction (PCR) machine in a multiplex format (multi-target simultaneous detection) with following identification of minimal time period required for detection of any airborne microbial targets in the ambient air. The experimental program was undertaken under controlled laboratory conditions utilizing of up to 7 plex PCR format for detection of airborne microbial targets in the dynamic aerosol chamber. The technology demonstrated efficient operation and was capable of detecting airborne microbial targets during even shortest experimental sampling periods of 10 s. The total time of detection/analysis was less than 40 min. The proposed sampling timeframe represents realistic scenario of bioaerosol exposure of humans’ moving through areas potentially contaminated by bioaerosol originated from natural or anthropogenic sources. The research outcomes look very promising enabling development of new generation of portable, reliable and fast bioaerosol monitors, which are unique and highly demanded in the areas of defence, public health and others.