In the past decades there has been a dramatic increase in the number of opportunistic fungal infections. Establishing the diagnosis of opportunistic fungal infections in compromised patients is not simple. The laboratory diagnostic tests include microscopic examination, culture and serological tests. Although the most reliable method is the histologic examination, various opportunistic fungal agents can reveal similar histologic morphology. Culture should be attempted, however, the isolation of these organisms from cultures must be interpretated with caution, because the causing agents for opportunistic fungal infections are common laboratory contaminants. Serology for fungal infections has limited value except cryptococcal antigen: the usefulness of detection of antigenemia in invasive candidiasis and invasive aspergillosis has been limited by the rapid clearance of Candida mannan and Aspergillus galactomann from serum, which results in only moderate sensitivity for the disease. Therefore, it should be appreciated that every laboratory test, for the diagnosis of opportunistic infections, has its limitations and should be interpreted with caution.