Fungal infections are a diagnostic and therapeutic problem of increasing concern due to the frequency and severity of disseminated infection in immunocompromised patients. Culture-based methods are characteristically slow and have poor sensitivity; hence, other methods, based on the detection of fungus-specific genetic, antigenic and metabolic components are being developed to enable early diagnosis and specific treatment. Moreover, reproducible antifungal susceptibility methods that can be adapted for use in clinical laboratories have been standardized to allow in vitro detection of resistance, which correlates with a less favorable clinical outcome. In this paper we review the main microbiological procedures available for the diagnosis of fungal infections and for antifungal susceptibility testing.