Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs . Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated . In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea . Moreover, the Δ dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum , and reduced production of 62 secondary metabolites (SM) including yellow‐coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea Δ dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea , and 11 was upregulated in the Δ dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B . cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δ dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology.