Temporin B (TB) is a 13 amino acid long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We could recently show that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low µM concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions showing a preference for anionic lipids and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using FITC-labelled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy independent manner. Peptide treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures were disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that the TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and promises its great potential as a future anti-Candida therapeutic.