This study expands the knowledge on chemical synthesis and properties of Hb40-61a as well as provides results of the first steps given towards knowing how it kills Candida cells. For the first time, this peptide, its all-D analogue (D-Hb40-61a) and its fluorescently labeled analogue (FAM-Hb40-61a) were successfully assembled on resin at 60 °C using conventional heating in all steps. Purified and characterized, these peptides exhibited very low toxicity on human erythrocytes. Hb40-61a and D-Hb40-61a were equally active against Candida strains, ruling out sterically specific interactions on their working mechanism. Cell permeabilization assays confirmed progressive damage of the yeast plasma membrane with increasing concentrations of Hb40-61a. While experiment using the fluorescent probe DiBAC4(5) revealed that this synthetic hemocidin alters the yeast plasma membrane potential, test employing DPH indicated that Hb40-61a might affect its dynamics. Exposure of the yeast cells to FAM-Hb40-61a showed that the peptide accumulates in the cell membrane at the 1/2 MIC, but stains about 97% of the cells at the MIC. Such effect is salt-dependent and partially energy-dependent. These new findings indicate that the central target of Hb40-61a in Candida cells is the plasma membrane and that this synthetic hemocidin should be considered as a potential candidacidal for topic uses.