<p>Cliotides are cyclic peptides reported in <i>Clitoria ternatea</i>, and belong to the class of cyclotides. Cliotides are ultrastable due to their cyclic cystine knot (CCK) motif and possess biological activities, including antibacterial, pesticidal, hemolytic and anticancer properties. In the present study, in silico analysis of 69 cliotides was performed for understanding their sequence diversity, physicochemical properties, phylogeny, bioactivity and to evaluate potent antibacterial cliotides based on physiochemical parameters and membrane interaction studies. Of the 69 cliotides, only 17 have been screened for bioactivities, with 15 cliotides experimentally tested for antibacterial properties. These 15 cliotides were classified into potent and non-potent groups based on their Minimum Inhibitory Concentration (MIC) values. Analysis of physicochemical parameters suggests that cliotides with higher net charge, isoelectric point and, number of basic amino acids exhibit potent antibacterial activity. Further, these three physicochemical properties are shown to have positive correlation. Membrane-cliotide interactions revealed that potent antibacterial cliotides (cliotide T1, cliotide T4) had lower ΔG and deeper membrane penetration than non-potent ones (cliotide T2, cliotide T3). The potent cliotides exhibited more hydrophobic amino acids with larger hydrophobic surface than non-potent ones. The analysis of surface structure models of these cliotides aligned with membrane-peptide studies. Thus, in silico studies can aid in identification of cliotides with potent antibacterial activity for prioritizing their in vitro/in vivo investigations, with a therapeutic perspective.</p>