The incidence of invasive infections caused by fungi has been increasing for the last few decades. This is primarily due to the significant increases in the populations of at-risk patients; this includes those receiving immunosuppressive chemotherapy for various malignancies, solid organ transplant recipients and those receiving prolonged steroid treatment. New diagnostic approaches have been developed based on non-culture-based methods, which may allow early diagnosis and treatment of fungal infections. Laboratory procedures in diagnostic mycology are directed mainly toward the direct demonstration of the pathogenic fungi in clinical specimens by microscopy along with successful isolation of pathogenic fungi by using various culture techniques. They also help in the prediction of possible therapeutic outcome by determining antifungal susceptibility and can also be used in epidemiological studies by tracing the source of infection. The purpose of our review is to provide the reader with comprehensive and up-to-date information on diagnostic methods.