ABSTRACTArmillaria ostoyae, a species among the destructive forest pathogens from the genusArmillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate,Trichoderma atrovirideSZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploidA. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA inin vitrodual culture assays to test the molecular arsenal ofTrichodermaantagonism and the defense mechanisms ofArmillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms.IMPORTANCEArmillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study foundTrichoderma atrovirideto be highly effective in controllingArmillariagrowth; therefore, our current work explored the molecular mechanisms that might play a key role inTrichoderma-Armillariainteractions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploidArmillariaisolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved inArmillariadefense againstTrichodermaand the genes potentially involved in the efficiency ofTrichodermato controlArmillaria. In addition, using a sensitive haploidArmillariastrain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses ofArmillaria ostoyaetoward diverseTrichodermaisolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.