The RNAi machinery is generally involved in genome protection in filamentous fungi; however, the physiological role of RNAi has been poorly studied in fungal models. Here, we report that in the filamentous fungusTrichoderma atroviride, the products of thedcr2andrdr3genes control reproductive development, because mutations in these genes affect conidiation. In addition, Dcr1 together with Dcr2 control vegetative growth since Δdcr1, Δdcr2andΔdcr1Δdcr2present morphological alterations. Whole-genome transcriptional analysis of WT, Δdcr1, Δdcr2andΔdcr1Δdcr2show that each Dicer controls different biological processes, such as development or metabolism, which could explain the lack of conidiation in the mutants. Finally, we observed sRNAs that are differentially expressed in the WT and Δdcr2. The expression of some of these sRNAs correlates with the expression of differential transcripts, suggesting that these mRNAs may contain the corresponding targets. Together these data show that inT. atroviride, the RNAi machinery plays a central role in endogenous processes such as development and fitness, beyond controlling genome protection against invasive nucleic acids as reported for other fungi.