Recommendations were developed using the Grading of Recommendations Assessment, Development, and Evaluation approach.<b>Results:</b> This guideline includes specific recommendations on the use of galactomannan testing in serum and BAL and for the diagnosis of invasive pulmonary aspergillosis, the role of PCR in the diagnosis of invasive pulmonary aspergillosis, the role of β-d-glucan assays in the diagnosis of invasive candidiasis, and the application of serology and antigen testing in the diagnosis of the endemic mycoses.<b>Conclusions:</b> Rapid, accurate diagnosis of fungal infections relies on appropriate application of laboratory testing, including antigen testing, serological testing, and PCR-based assays.