An increasing number of patients are undergoing transplantation procedures or receiving aggressive immunosuppression and chemotherapy. The growing population of immunocompromised hosts has led to a rise in the prevalence of invasive fungal infections due to yeasts and molds. The introduction of new antifungal agents and recent reports of resistance emerging during treatment of fungal infections have highlighted the need for in vitro susceptibility testing. Various testing procedures have been proposed, including macrodilution and microdilution, agar diffusion, disk diffusion, and Etest (AB Biodisk, Solna, Sweden). Establishing clinical correlation with antifungal susceptibility testing, however, is a huge challenge because susceptibility techniques do not take into account the dynamic and complex biology of fungi exposed to an antifungal in vivo. This paper reviews the available methods for antifungal susceptibility testing of yeasts and filamentous fungi and the data regarding the clinical implications of in vitro testing.