Invasive fungal infections have become a major cause of morbidity and mortality over the past 3 decades. Organ transplantation, the use of aggressive chemotherapy, and the availability and widespread use of immunosuppressive treatments for many medical ailments have resulted in large populations of patients who are at risk for fungal disease. Early diagnosis and prompt therapy are instrumental to successful treatment of these infections, but conventional methods for diagnosis of fungal disease are slow and lack sensitivity. Important advances in diagnosing invasive mycoses, particularly in laboratory-based testing, have been realized over the years. Antigen-based assays, new laboratory methods for identification of fungi, and reference guidelines for susceptibility testing have been developed and validated for use in clinical laboratories. We review these technological advances and our understanding of their clinical application and impact.