The increasing number of resistant bacteria is stimulating the search for alternative antibiotics. Antimicrobial peptides pose an interesting alternative as they are actively used by every species as part of their host defence mechanisms. Gomesin, a 18-residues peptide with two disulfide bonds arranged with ladder connection, is produced in hemocytes of the Brazilian spider Acanthoscurria gomesiana and was shown to have potent antimicrobial activity towards Gram-negative bacteria and selective anticancer properties against melanoma cells. In a recent study, a cyclic analogue of Gomesin was shown to be as active but more stable than its native form. In the current study, we rationally designed a series of cyclic Gomesin analogues to improve its antimicrobial activity and investigate its mode-of-action. We successfully improved the activity by ~10-fold against tested Gram negative and Gram positive bacteria and melanoma cells without increasing toxicity towards red blood cells. Mode-of-action studies revealed that cyclic Gomesin and its analogues act on bacteria and melanoma cells by targeting and disrupting cell membranes. Interestingly, cyclic Gomesin analogues were found to enter inside other tested human cell lines in a membrane-dependent but non-disruptive way suggesting that membrane-disruption vs membrane-permeation is cell-type dependent and is very likely determined by cell membrane composition. In summary, we have designed stable cyclic Gomesin analogues with potent antimicrobial activity and with efficient cell-penetrating properties that can be used as stable scaffolds to deliver active sequences inside human cells. In addition, we have found that their mode-of-action and anticancer properties are dependent on the composition of the cell membrane.