Background <p>Environmental air sampling holds significant potential as a tool for viral surveillance. Its use in agricultural and indoor settings has demonstrated its feasibility and effectiveness but despite this, it has rarely been used in wildlife settings.</p> Methods <p>To enable future applications, we optimized key parameters in air sampling methodology using a cave-roosting bat assemblage as a model system. We systematically investigated the impact of sampling conditions (flow rate, sampling duration, and sampling location/deployment time) and post-sampling treatments (DNA/RNA Shield ratios and secondary filtration) on three viral metrics – total mammalian virus abundance, mammalian RNA virus abundance, and Shannon diversity index – generated from next-generation sequencing data.</p> Results <p>We first showed that air sampling can recover broad viral diversity, including alphacoronaviruses and betacoronaviruses. The sampling conditions for maximizing viral metrics were larger air sample volumes (≥24,000 liters) and sampling inside the cave while the bats were roosting, as opposed to at the cave entrance during emergence. Post-sampling treatments had limited impact on viral metrics, but their application may vary depending on the objectives of the study. </p> Conclusion <p>This work provides a proof-of-concept for applying air sampling for wildlife viral surveillance in a cave-roosting bat assemblage and identifies key sampling parameters.</p>