Candida albicans is the principal opportunistic fungal pathogen in nosocomial settings and resistance to antifungal drugs is on the rise. Antimicrobial peptides from natural sources are promising novel therapeutics against C. albicans. OsDef2 defensin was previously found to be active against only Gram-positive bacteria, whereas derived fragments Os and its cysteine-free analogue, Os-C, are active against Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, OsDef2-derived analogues and fragments were screened for anticandidal activity with the aim to identify peptides with antifungal activity and in so doing obtain a better understanding of the structural requirements for activity and modes of action. Os, Os-C and Os(11-22)NH2 , a Os-truncated carboxy-terminal-amidated fragment, had the most significant antifungal activities, with minimum fungicidal concentrations (MFCs) in the micromolar range (6-28 μM). C. albicans killing was rapid and occurred within 30-60 min. Further investigations showed all three peptides interacted with cell wall derived polysaccharides while both Os and Os(11-22)NH2 permeabilized fungal liposomes. Confocal laser scanning microscopy confirmed that Os-C and Os(11-22)NH2 could enter the cytosol of live cells and subsequent findings suggest that the uptake of Os and Os-C, in contrast to Os(11-22)NH2 , is energy dependent. Although Os, Os-C and Os(11-22)NH2 induced the production of reactive oxygen species (ROS), co-incubation with ascorbic acid revealed that only ROS generated by Os-C and to a lesser extent Os(11-22)NH2 resulted in cell death. Overall, Os, Os-C and Os(11-22)NH2 are promising candidacidal agents.