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The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH₂) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis …