species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism, spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host's cell wall. Here, we explored the participation of the RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the -RNAi when interacting with AG2 and AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the -host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition ( ) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established.IMPORTANCEThere is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of species, there are still unsolved questions related to how regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by .