Violaceous plant Hybanthus enneaspermus F.Muell is a medicinal herb with diverse bioactivities and is commonly used by the Indian community as a natural libido enhancer. Violaceae plant family is a rich source of cyclotides. In this study, ten novel cyclotide sequences and one known cyclotide (cycloviolacin O2), were identified and isolated from this plant, and were sequenced using tandem mass spectroscopy. Among these peptides, six of them were confirmed by NMR spectroscopy and showed similar overall structures to either cycloviolacin O2 or O1. Their bioactivities were examined on cells cytotoxicity and hemolytic assays. Surface plasmon resonance technique was also used to further characterize their lipid binding properties. A strong binding preference to phosphatidylethanolamine headgroup phospholipid was observed and correlates well to their cytotoxicity profiles. Overall, this study helps broaden the knowledge of indigenous Indian herb H. enneaspermus, expands the cyclotides sequence diversity and provides scaffold options for grafting studies in future.