We are actively exploring the use of synthetic antifungal peptides (AFPs) as novel pharmaceuticals to treat human fungal infections. Our recent studies have focused on the mode-of-action of various small rationally designed AFPs and synthetic AFPs derived from plant defensins. For this purpose we are using the fungal model Neurospora crassa and the human pathogen Aspergillus fumigatus combined with live-cell imaging of fluorescently labelled AFPs and other live-cell probes, inhibitor treatments and mutant analyses. PAF26 is a de novo-designed hexapeptide possessing two well-defined motifs: N-terminal cationic and C-terminal hydrophobic regions. We have characterised how each motif is responsible for PAF26’s dynamic antifungal mechanism of action involving the electrostatic interaction with cells, cellular internalisation, and cell killing. PAF26 increases cytosolic free Ca2+ ([Ca2+]c) and several Ca2+ signalling/homeostatic mutants are resistant to the AFP. Different peptide sequences derived from the γ-core motifs of MsDef1 and MtDef4 defensins inhibit conidial germination and hyphal fusion, and influence [Ca2+]c, with different potencies and specificities. Our results provide new mechanistic insights into the mode-of-action of AFPs that should help us design new synthetic AFP-based drugs with improved activity and stability.