This review will address the value of nucleic acid amplification techniques used for the laboratory diagnosis of fungal infections. Although detection of all fungi will be considered, the emphasis will be placed on diagnosis of disseminated candidiasis. The diagnosis of most serious life threatening fungal infections in immunosuppressed patients remains a laboratory dilemma. The antemortem diagnosis of disseminated candidiasis, if made at all, is often made just prior to the death of the patient negating any hope of successful medical/surgical intervention. Molecular techniques such as those based on the polymerase chain reaction offer increased sensitivity since, in theory, these techniques require the presence of a single fungal cell. This manuscript will review 1) the current problems related to classical methods of diagnosing life threatening fungal infections; 2) considerations one must address relative to use of nucleic acid-based approaches such as polymerase chain reaction (PCR) for diagnosis of these fungal infections; and 3) status of use of these molecular approaches in the clinical mycology laboratory at the present time.