Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance.