Updated publication reference for PubMed record(s): 32414842. Cyclotides are a family of plant-derived peptides characterized by a ~30 amino acid cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and in particular their cytotoxicity has attracted significant attention for its potential anti-cancer applications. Hybanthus enneaspermus is a medicinal herb widely used in India as a natural libido enhancer, and a previous study indicated that it may contain cyclotides. In the current study, 11 novel cyclotides and one known cyclotide (cycloviolacin O2) were identified and isolated from H. enneaspermus. Their sequences were determined using tandem MS, and NMR analysis of six of these cyclotides indicates their three-dimensional structures are similar to those of other known cyclotides. The most abundant cyclotide in this plant, hyen D, has comparable cytotoxicity against cancer cells to cycloviolacin O2, the most cytotoxic cyclotide discovered so far. Herein we provide a mechanistic insight into the mode-of-action of the novel cyclotides’ cytotoxicity. Hyen D, E, L, M, and cycloviolacin O2 preferentially interacted with model lipid membranes containing phospholipids with phosphatidylethanolamine-headgroups, as measured by surface plasmon resonance. Cyclotide-membrane interactions correlated with cytotoxicity, suggesting that membrane binding underpins cytotoxicity. Overall, this study helps broaden knowledge on the indigenous Indian herb H. enneaspermus, and reports the discovery of novel cyclotides with potent anti-cancer activity.