RNA interference (RNAi) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding RNAs (sRNAs) of about 20 to 30 nucleotides. Its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. Initially described and characterized in Neurospora crassa, the RNAi is widespread in fungi, suggesting that it plays important functions in the fungal kingdom. Several RNAi-related mechanisms for maintenance of genome integrity, particularly protection against exogenous nucleic acids such as mobile elements, have been described in several fungi, suggesting that this is the main function of RNAi in the fungal kingdom. However, an increasing number of fungal sRNAs with regulatory functions generated by specific RNAi pathways have been identified. Several mechanistic aspects of the biogenesis of these sRNAs are known, but their function in fungal development and physiology is scarce, except for remarkable examples such as Mucor circinelloides, in which specific sRNAs clearly regulate responses to environmental and endogenous signals. Despite the retention of RNAi in most species, some fungal groups and species lack an active RNAi mechanism, suggesting that its loss may provide some selective advantage. This article summarizes the current understanding of RNAi functions in the fungal kingdom.