Invasive aspergillosis and other invasive fungal infections are associated with significant morbidity and mortality in immunocompromised patients, in large part due to limitations of existing diagnostic methods for these infections. Detection of species-specific volatile sesquiterpene metabolites of fungal origin in the breath of patients with invasive fungal infections allows the diagnosis and monitoring of these infections in vivo, non-invasively and more rapidly than possible with current diagnostic methods. While detection of exogenous microbial volatile metabolites in the breath has opened up a new and exciting dimension of diagnostic research and development in infectious diseases, we discuss the daunting challenges to volatile diagnostic biomarker discovery and clinical development.