Fungal infections have become a serious global health concern, made worse by limited antifungal therapies and an ever-increasing number of drug-resistant strains. Mass spectrometry (MS)-based proteomics offers a transformative approach towards apprehending fungal pathogenesis by enabling high-resolution mapping of protein expression, post-translational modifications, and host–pathogen interactions. This chapter discusses key proteomic strategies, including bottom-up and top-down workflows, and how they help in studying fungal virulence factors, stress responses, and immune evasion mechanisms. It further highlights advances in our ability to conduct a dual-proteome analysis that helps unravel the dynamic crosstalk between fungi and the host. Beyond pathogenesis, this approach has been accelerating antifungal drug discovery by identifying novel therapeutic targets, characterizing resistance pathways, and guiding biomarker development for early diagnosis. While technical and biological challenges remain, the integration of proteomics with advanced infection models, bioinformatics, and systems biology promises to revolutionize fungal diagnostics and therapeutics.