<p>Cyclotides are a class of plant-derived cyclic peptides having a distinctive structure with a cyclic cystine knot (CCK) motif. They are stable molecules that naturally play a role in plant defense. Till date, more than 750 cyclotides have been reported among diverse plant taxa belonging to Cucurbitaceae, Violaceae, Rubiaceae, Solanaceae, and Fabaceae. These native cyclotides exhibit several bioactivities, such as anti-bacterial, anti-HIV, anti-fungal, pesticidal, cytotoxic, and hemolytic activities which have immense significance in agriculture and therapeutics. The general mode of action of cyclotides is related to their structure, where their hydrophobic face penetrates the cell membrane and disrupts it to exhibit anti-microbial, cytotoxic, or hemolytic activities. Thus, the structure–activity relationship is of significance in cyclotides. Further, owing to their, small size, stability, and potential to interact and cross the membrane barrier of cells, they make promising choices for developing peptide-based biologics. However, challenges, such as production complexity, pharmacokinetic limitations, and off-target effects hinder their development. Advancements in cyclotide engineering, such as peptide grafting, ligand conjugation, and nanocarrier integration, heterologous production along with computational design optimization, can help overcome these challenges. Given the potential of these cyclic peptides, the present review focuses on the diversity, bioactivities, and structure–activity relationships of cyclotides, and&#xa0;advancements in cyclotides engineering emphasizing their unique attributes for diverse medical and biotechnological applications.</p> Graphical abstract <p></p>