This study sought to investigate the antibacterial activity and mode of action of two antimicrobial peptides, Os and Os-C, derived from a soft tick defensin against Staphylococcus epidermidis. The antibacterial activity of both peptides was investigated in 1% tryptic soy broth medium and Os and Os-C had a minimum inhibitory concentration of 3.43 µM and 10 µM, respectively. Both peptides were able to cause 99.5% killing at 10 µM following three hours of treatment. The mode of action of the two peptides was also investigated using the Live/Dead membrane permeability kit in addition to a membrane potential sensitive dye, DiBAC₄(3). It was shown that both melittin and Os caused membrane permeabilisation at 0.4 µM and 7 µM, respectively, whereas Os-C did not cause permeabilisation at 20 µM. Os and Os-C were further shown to affect membrane potential by causing membrane depolarisation. Further visualisation of the cells using scanning and transmission electron microscopy revealed that both Os and Os-C including melittin adversely affected the S. epidermidis membrane causing membrane blebbing, indentation with Os and melittin inducing ghost cell formation. Transmission electron microscopy further revealed that Os was lytic whereas Os-C induced mesosome formation, a possible consequence of reactive oxygen species production. Both peptides were thus active against the pathogen with further development necessary for increased activity and stability of Os and Os-C against S. epidermidis.