Fungal biofilm-related infections, most notably those caused by the Candida and Aspergillus genera, need to be diagnosed accurately and rapidly to avoid often unfavorable outcomes. Despite diagnosis of these infections is still based on the traditional histopathology and culture, the use of newer, rapid methods has enormously enhanced the diagnostic capability of a modern clinical mycology laboratory. Thus, while accurate species-level identification of fungal isolates can be achieved with turnaround times considerably shortened, nucleic acid-based or antigen-based detection methods can be considered useful adjuncts for the diagnosis of invasive forms of candidiasis and aspergillosis. Furthermore, simple, reproducible, and fast methods have been developed to quantify biofilm production by fungal isolates in vitro. In this end, isolates can be categorized as low, moderate, or high biofilm-forming, and this categorization may reflect their differential response to the conventional antifungal therapy. By means of drug susceptibility testing performed on fungal biofilm-growing isolates, it is now possible to evaluate not only the activity of conventional antifungal agents, but also of novel anti-biofilm agents. Despite this, future diagnostic methods need to target specific biofilm components/molecules, in order to provide a direct proof of the presence of this growth phenotype on the site of infection. In the meantime, our knowledge of the processes underlying the adaptive drug resistance within the biofilm has put into evidence biofilm-specific molecules that could be potentially helpful as therapeutic targets. Surely, the successful management of clinically relevant fungal biofilms will rely upon the advancement and/or refinement of these approaches.