Cyclotide proteins are the largest and most well-known family of cyclic proteins. These proteins have disulfide-rich bonds with 30 amino acid residues comprising C and N terminals to induce cyclization. They are found in various plant species, such as the Violaceae family. Bioactive peptides possess CCK (cyclic cysteine knots), which contain 28 to 37 amino acids and are useful in biotechnology and for producing peptide-based drugs. The Violaceae family is a distinct plant family that expresses the conserved cyclotide genes. In this study, we investigated the 14 species of the Violaceae family to show their antimicrobial activity against the two bacterial species, Escherichia coli and Staphylococcus aureus. Cyclotide genes were isolated from 5 species of V. tricolor through PCR screening. The phylogenetic analysis revealed that viola tricolor (white blotch), viola tricolor (Rose Blotch), viola tricolor (purple blotch), viola tricolor (rosalyne) and viola tricolor (tigerary) showed the homology with the other species of the Violaceae family. The discovery of the cyclotide precursor gene from the Violaceae plant species defined their structural sequence and evolutionary link to their diversity from other species. It opened a new research line on the evolution of cyclotide proteins. This research will foster the production of new drugs that interact with only pathogens, not humans, and to develop, produce, transport or access cheaper medications. The present research will also contribute to future exploration in agriculture.