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• We propose a wind tunnel-based method to comprehensively evaluate the performance of bioaerosol samplers at 2, 8, and 20 km/h. • Performance of bioaerosol samplers was evaluated based on several parameters. • SASS 2300 and BSA-350 are appropriate choices for qualitative analysis and isolation of microorganisms from ambient air. • Polycarbonate and gelatin filters are recommended for long-term sampling and quantitative analyses of bioaerosols without microbial viability requirements. • AGI-30 and BioSampler are suitable for short-term sampling and quantitative analyses of bioaerosols in laboratory environments. Choosing a suitable bioaerosol sampler for atmospheric microbial monitoring has been a challenge to researchers interested in environmental microbiology, especially during a pandemic. However, a comprehensive and integrated evaluation method to fully assess bioaerosol sampler performance is still lacking. Herein, we constructed a customized wind tunnel operated at 2–20 km/h wind speed to systematically and efficiently evaluate the performance of six frequently used samplers, where various aerosols, including Arizona test dust, bacterial spores, gram-positive and gram-negative bacteria, phages, and viruses, were generated. After 10 or 60 min of sampling, the physical and biological sampling efficiency and short or long-term sampling capabilities were determined by performing aerodynamic particle size analysis, live microbial culturing, and a qPCR assay. The results showed that AGI-30 and BioSampler impingers have good physical and biological sampling efficiencies for short-term sampling. However, their ability to capture aerosols at low concentrations is restricted. SASS 2300 and BSA-350 wet-wall cyclones had excellent enrichment ratios and high microbial cultivability in both short-term and long-term sampling; however, they were not suitable for quantitative studies of aerosols. Polycarbonate filter samplers showed outstanding performance in physical and long-term sampling but lacked the ability to maintain microbial activity, which can be improved by gelatin filter samplers. However, limitations remain for some fragile microorganisms, such as E. coli phage PhiX174 and coronavirus GX_P2V. In addition, the effects of wind speed and direction should be considered when sampling particles larger than 4 µm. This study provides an improved strategy and guidance for the characterization and selection of a bioaerosol sampler for better measurement and interpretation of collected ambient bioaerosols. [ABSTRACT FROM AUTHOR]