RNA-based medicines are an emerging class of drugs that are changing the way we treat infectious diseases. The versatility of this class is clearly on display in the rapid design and adaptation of the mRNA vaccines to SARS-CoV-2. Despite the success of these approaches against viruses, research into RNA-based therapeutics to treat human fungal pathogens, a major source of human morbidity and mortality, is lacking. Here, we provide an improved mechanistic description of the RNA interference pathway in Aspergillus fumigatus and assess this pathway as a potential target for RNA-based medicines. We describe the genetic variation in RNA interference-associated genes in a large collection of environmental and clinical genomes, reveal the proteins regulated by the RNA interference system using advanced proteomics analysis, and define the components essential for hairpin-induced silencing. We then exploit this pathway using a heterologously expressed hairpin RNA construct to silence the pabA gene of A. fumigatus to inhibit growth. The data presented here provide a foundation for a mechanistic description of novel RNA regulatory pathways in A. fumigatus and reaffirm the importance of improved fungal RNA delivery mechanisms for the future design of RNA-based therapeutics against this important human fungal pathogen.