Many fungal genomes have now been sequenced and thousands of genes are being discovered. Gene disruption or inactivation technology offers an important tool not only for elucidating the function of the many unknown genes but also for the identification of genes essential for fungal growth and pathogenesis. A variety of gene-silencing methods that inhibit genes at the post-transcriptional level are now being used in both non-pathogenic and human pathogenic fungi. We focus on the recent advances in RNA-mediated gene silencing technologies and their potential for functional genomics studies in fungi.