Classically, diagnosis of fungal infections is based on microscopic examination coupled with attempts to culture the responsible fungus from a clinical sample. For some fungal infections (such as dermatophyte infections, infections with dimorphic fungi, and blood stream infections with Fusarium, Acremonium and allied genera) recovery of the fungus in culture from a patient with clinical symptoms is sufficient for diagnosis. However, in many cases, obtaining a yeast or filamentous fungus in culture is not easily interpreted in isolation. In such circumstances, decisions regarding the clinical significance of an isolate must consider the nature of the organism and the quantity isolated, the likelihood of it accidentally contaminating the specimen, whether fungal elements were seen upon microscopic examination of the sample, the clinical status of the patient, and whether there are other clinical or biological markers suggesting infection. This review discusses these considerations for the different types of clinical samples encountered in a microbiology laboratory.