The current increase in the number and significance of fungal infections, the expanding armamentarium of antifungal agents, and the emergence of the problem of antifungal drug resistance have been intensifying the importance of antifungal susceptibility testing (AST). The Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) in the United States and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) published standard methodologies in order to achieve higher reproducibility and allow direct inter-laboratory comparison of the susceptibility results. Nevertheless, several problems remain unresolved and the methods depend on long incubation periods of a minimum of 24 h (EUCAST) or even 48 h (CLSI). Over the last 15 years, successful applications of flow cytometric techniques to AST of both yeast and moulds have been reported. These techniques are based on the analysis of a great number of fungal cells individually and frequently rely on short incubation times of no more than a few hours. Considering these attributes, flow cytometry (FC) seems to have the potential to achieve clinical usefulness in the near future. The collection of data on the reproducibility of the results and on the correlation with clinical outcomes has barely started, however. Practical validation of the experimental methodologies is not granted before a significant amount of data addressing those questions is available.