The rise in fungal infections, driven by pathogens resistant to the limited scope of antifungal agents available, poses an increasing threat to global health and the economy. Addressing this challenge requires a thorough understanding of the mechanisms of antifungal agents and the development of advanced resistance diagnostic methods. This opinion manuscript highlights recent advancements in antifungal research, with a focus on chemical biology approaches, particularly the development of fluorescent probes derived from various antifungal agents. These probes reveal new aspects of antifungal activity and provide deeper insights into modes of action and resistance mechanisms. Live cell imaging of fungal pathogens labeled with these probes has uncovered novel strategies to enhance antifungal efficacy, understand virulence factors, and detect resistance. These unique small-molecule tools offer powerful new avenues for addressing the fungal infections crisis, harnessing chemical biology approaches to develop innovative solutions to the global challenges posed by fungi.