Air is a medium for dispersal of environmental DNA (eDNA) carried in bioaerosols, yet the atmosphere is mostly unexplored as a source of genetic material encompassing all domains of life. In this study, we designed and deployed a robust, sterilizable hardware system for airborne nucleic acid capture featuring active filtration of a quantifiable, controllable volume of air and a high-integrity chamber to protect the sample from loss or contamination. We used our hardware system on an aircraft across multiple height transects over major aerosolization sources to collect air eDNA, coupled with high-throughput amplicon sequencing using multiple DNA metabarcoding markers targeting bacteria, plants, and vertebrates to test the hypothesis of large-scale genetic presence of these bioaerosols throughout the planetary boundary layer in the lower troposphere. Here, we demonstrate that the multi-taxa DNA assemblages inventoried up to 2,500 m using our airplane-mounted hardware system are reflective of major aerosolization sources in the survey area and show previously unreported airborne species detections (i.e., Allium sativum L). We also pioneer an aerial survey flight grid standardized for atmospheric sampling of genetic material and aeroallergens using a light aircraft and limited resources. Our results show that air eDNA from terrestrial bacteria, plants, and vertebrates is detectable up to high altitude using our airborne air sampler and demonstrate the usefulness of light aircraft in monitoring campaigns. However, our work also underscores the need for improved marker choices and reference databases for species in the air column, particularly eukaryotes. Taken together, our findings reveal strong connectivity or mixing of terrestrial-associated eDNA from ground level aerosolization sources and the atmosphere, and we recommend that parameters and indices considering lifting action, atmospheric instability, and potential for convection be incorporated in future surveys for air eDNA. Overall, this work establishes a foundation for light aircraft campaigns to comprehensively and economically inventory bioaerosol emissions and impacts at scale, enabling transformative future opportunities in airborne DNA technology.