Fungal infections were spotlighted by the WHO in 2022 as being an important medical challenge of the 21st century, causing up to 3.75 million deaths among 1 billion infections annually and case-fatality rates of up to 90% in some populations. Immunocompromised individuals are especially susceptible to fungal infections, which are caused by opportunistic pathogens like Aspergillus fumigatus. Due to shortcomings in treatment and early diagnosis as well as an increased emergence of antifungal resistance, new diagnostic and therapeutic strategies are essential. RNA therapeutics are a potentially beneficial approach that might emerge from the expanding field of extracellular RNAs (exRNA), where a variety of RNA species were demonstrated to mediate cross-kingdom communication in fungal infection contexts of plants, insects, and mammalian immune cells. These exRNAs can be released in association with RNA binding proteins (RBPs) or extracellular vesicles (EVs), of which the latter derived from A. fumigatus-infected polymorphonuclear leukocytes (PMNs), were shown to bear antifungal capacity. To uncover a possible involvement of exRNAs in antifungal defense, we investigated RNA isolates from large and small EV fractions alongside small RNA-sequencing. Utilizing capillary electrophoresis, the extracellular environment was revealed to be enriched for 20-200 nt small RNAs (sRNA), which according to sRNA-sequencing displayed high abundances of miRNAs. Surprisingly, these miRNAs were enriched for specific, conserved candidates with known antimicrobial properties and potentially play a gene modulatory role in the fungus in cooperation with fungal or human RNA interference (RNAi) effectors. Subsequent investigations focused on the characterization of the identified miRNAs, involvement of key RNAi components, and definition of their role in A. fumigatus defense. We hope that these findings will contribute to the foundation of the exRNAs as a mediator of host-pathogen interactions and benefit development of new RNA-based diagnostics and therapeutics against this important human pathogen in the future.