In this study the structural characteristics and antibacterial activities of two peptides derived from the carboxy-terminal of a tick defensin were investigated. Two defensin isoforms (OsDef1 and OsDef2) were previously identified in the midgut of the tick, Ornithodoros savignyi. Both OsDef1 and OsDef2 were found to be active against Gram-positive bacteria but showed no antibacterial activity against Gram-negative bacteria. OsDef2 was found to be slightly more active than OsDef1 and was, therefore, used as the template for the design of smaller antimicrobial peptides. Two peptide analogues were synthesised using the carboxy-terminal sequence of OsDef2 and differed in that in the one peptide the cysteine residues were present (Os) and in the other the cysteine residues were omitted (Os-C). Structurally, Os contained more α-helical properties than Os-C, whereas Os-C was more β-sheeted when prepared in 25 mM SDS. Both Os and Os-C showed no antibacterial activity when tested in Luria-Bertani broth or Mueller-Hinton broth indicating that the activities of Os and Os-C were influenced by the presence of broth salts and proteins. When tested in sodium phosphate buffer, both Os and Os-C exhibited Gram-positive and Gram-negative antibacterial activity. Os was slightly more active than Os-C against 3 of the 4 tested strains, with minimum bactericidal concentrations (MBCs) ranging from 0.94 μg/ml to 3.75 μg/ml. Os retained bactericidal activity against both Staphylococcus aureus and Escherichia coli when tested in the presence of 100 mM NaCl or 30% human serum. Os-C retained activity against E. coli in the presence of NaCl but became inactive in 30% human serum against both bacterial strains. At the MBCs, Os exhibited faster killing kinetics than Os-C killing both Bacillus subtilis and E. coli within 5 min, whereas Os-C took up to 120 min and 60 min, respectively. SYTOX Green permeabilization assays showed that both Os and Os-C caused permeabilization of E. coli membranes after 30 min exposure. At high peptide concentrations, both Os and Os-C were shown to interact with plasmid DNA. Both Os and Os-C exhibited no cytotoxic effects against SC-1 and Caco-2 cell lines, even at peptide concentrations 32 times higher than the highest MBC.