Fusarium kuroshium and the ambrosia beetle Euwallacea kuroshio attack several tree species, including the arroyo willow (Salix lasiolepis). Despite the importance of this pest, there is no molecular information about this plant-fungus interaction. Using an in vitro pathosystem of S. lasiolepis leaves – F. kurosihum and the RNA-seq approach, this study analyzed for the first time the transcriptome of both organisms at 4-, 8-, and 12-days post-inoculation. De novo assembly was done to generate the willow transcriptome, and a total of 361,2151 unique transcripts (UniGenes) were found. 4,583 UniGenes responded to the infection, and the posterior analysis showed that it contains the molecular elements to develop both pattern- and effector-triggered immunity responses (PTI and ETI, respectively). Significant expression changes of genes involved in an oxidative burst, Ca2+ signaling, and mutually antagonistic interaction between salicylic acid- and jasmonic acid/ethylene-dependent signaling suggest that the willow recognized the fungus and triggered downstream responses. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense. Concerning F. kuroshium, 178 induced UniGenes that show homology with genes involved in the pathogenicity in other ascomycetes fungi plus transport proteins, ribosomal proteins, and uncharacterized proteins with specific domains were identified. Collectively these data provide a basis for future research on the molecular response of this transcendental tree during biotic stress, specifically during Fusarium dieback disease.