Introduction: The importance of the antitumor activity of some antimicrobial peptides (AMPs) is being increasingly recognized. The antimicrobial peptide, tachyplesin, has been shown to exhibit anticancer properties and a linear, cysteine deleted analogue (CDT), was found to retain its antibacterial function.Objectives: The objective was to test CDT and related analogues against normal mammalian, bacterial, and cancer cells to determine their effectiveness and then utilize specific assays to determine a possible mechanism of action. Methods: We used sequence reversal and D-amino acids to synthesize four CDT analogues by solid phase peptide synthesis. A number of assays were used including liposome dye-leakage, antibacterial activity against both Gram-positive and Gram-negative bacterial strains, hemolytic assays, methyl thiazolyl tetrazolium (MTT), and apoptosis to examine their effectiveness as both AMPs and anti-cancer peptides (ACPs). We then tested the analogues for their ability to inhibit proliferation of the human lung cancer cell line, A549. Results: We found that D-CDT exhibited the best bactericidal properties of those tested and was not damaging to red blood cells. Both D-CDT and reverse D-CDT showed a dose-dependent reduction of cell viability. However, D-CDT was most effective with an IC50 of 9.814 μM, a value 9-fold lower than that calculated for reverse D-CDT (90.16 μM). Apoptosis does not appear to be a mechanism by which D-CDT exerts its anticancer properties since > 100 μM was required to increase activation of caspase 3. Moreover, the ERK1/2 pathway is also unlikely since only a modest (20%) decrease of activity was observed with > 100 μM D-CDT. However, D-CDT was found to operate via a hyaluronan (HA)-dependent mechanism as pretreatment of the cells with hyaluronidase decreased the cytotoxic effects of D-CDT on A549 cells and increased its IC50 29-fold to 283.9 μM. Conclusion: D-CDT is both an effective AMP and ACP, and likely exerts its anticancer effects through both membranolytic as well as an HA-mediated mechanism.