A comprehensive review of diagnostic techniques for opportunistic systemic mycoses focused on invasive mould disease in immunocompromised patients is presented. We first analysed conventional diagnostic methods, such as microscopy examination, culture and radiology, underlining their limitations, which have led to the development of alternative methods, such as the detection of fungal components. Among these we highlight fungal antigen and DNA quantification, which make it possible to detect infections early and start appropriate treatment. We also briefly review the methods for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications. Furthermore, we analyse the recommendations for therapeutic drug monitoring of antifungal agents in body fluids.