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The ability to collect size-fractionated airborne particles that contain viable bacteria and fungi directly into liquid medium while also maintaining their viability is critical for assessing exposure risks. In this study, we present the BioCascade impactor, a novel device designed to collect airborne particles into liquid based on their aerodynamic diameter in three sequential stages (>9.74, 3.94–9.74, and 1.38–3.94 µm when operated at 8.5 L/min). Aerosol samples containing microorganisms—either Saccharomyces kudriavzevii or Micrococcus luteus, were used to evaluate the performance of the BioCascade (BC) paired with either the VIable Virus Aerosol Sampler (VIVAS) or a gelatin filter (GF) as stage 4 to collect particles <1.38 µm. Stages 2 and 3 collected the largest fractions of viable S. kudriavzevii when paired with VIVAS (0.468) and GF (0.519), respectively. Stage 3 collected the largest fraction of viable M. luteus particles in both BC + VIVAS (0.791) and BC + GF (0.950) configurations. The distribution function of viable microorganisms was consistent with the size distributions measured by the Aerodynamic Particle Sizer. Testing with both bioaerosol species confirmed no internal loss and no re-aerosolization occurred within the BC. Irrespective of the bioaerosol tested, stages 1, 3, and 4 maintained ≥ 80% of viability, while stage 2 maintained only 37% and 73% of viable S. kudriavzevii and M. luteus, respectively. The low viability that occurred in stage 2 warrants further investigation. Our work shows that the BC can efficiently size-classify and collect bioaerosols without re-aerosolization and effectively maintain the viability of collected microorganisms. Copyright © 2024 American Association for Aerosol Research [ABSTRACT FROM AUTHOR]