Wildland fire is a major producer of aerosols from combustion of vegetation and soils, but little is known about the abundance and composition of smoke’s biological content. Bioaerosols, or aerosols derived from biological sources, may be a significant component of the aerosol load vectored in wildland fire smoke. If bioaerosols are injected into the upper troposphere via high-intensity wildland fires and transported across continents, there may be consequences for the ecosystems they reach. Such transport would also alter the concept of a wildfire’s perimeter and the disturbance domain of its impact. Recent research has revealed that viable microorganisms are directly aerosolized during biomass combustion, but sampling systems and methodology for quantifying this phenomenon are poorly developed. Using a series of prescribed fires in frequently burned forest ecosystems, we report the results of employing a small rotary-wing unmanned aircraft system (UAS) to concurrently sample aerosolized bacteria and fungi, particulate matter, and micrometeorology in smoke plumes versus background conditions. Airborne impaction-based bioaerosol sampling indicated that microbial composition differed between background air and smoke, with seven unique organisms in smoke vs. three in background air. The air temperature was negatively correlated with the number of fungal colony-forming units detected. Our results demonstrate the utility of a UAS-based sampling platform for active sampling of viable aerosolized microbes in smoke arising from wildland fires. This methodology can be extended to sample viable microbes in a wide variety of emissions sampling pursuits, especially those in hazardous and inaccessible environments.