The occurrence and distribution of new and re-emerging fungal pathogens, along with rates of antifungal resistance, are rising across the globe, and correspondingly, so are our awareness and call for action to address this public health concern. To effectively detect, monitor, and treat fungal infections, biological insights into the mechanisms that regulate pathogenesis, influence survival, and promote resistance are urgently needed. Mass spectrometry-based proteomics is a high-resolution technique that enables the identification and quantification of proteins across diverse biological systems to better understand the biology driving phenotypes. In this review, we highlight dynamic and innovative applications of proteomics to characterize three critical fungal pathogens (