Despite the introduction of new antifungal agents, resistances to antifungal therapy continue to increase and outcome of invasive fungal infections treatment is frequently suboptimal. A large amount of the recent effort in antifungal drug discovery has focused on a limited set of targets with functions known or expected to be important for fungal viability and virulence. A variety of techniques can be used to identify fungal genes of interest. Gene expression profiling, RNA mediated gene silencing and insertional mutagenesis are three main molecular genetics technologies used to identify and validate antifungal drug targets. The term RNA interference (RNAi) refers to a cellular process by which a sequence-specific double-stranded RNA (dsRNA) inhibits the expression of a gene. This mechanism is strongly conserved in eukaryotes and has been documented to be existed in different fungal species such as Candida albicans, Aspergillus nidulans and Penicillium marneffei. Many vital and virulence genes have been successfully knocked down using RNAi technology. RNAi can be regarded as a promising approach for discovery of new gene targets for the design of fungus-specific antifungal agents. Here we discuss about a novel approach and its application in designing new molecular antifungal targets.