The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in . are a class of highly stable, cyclic, and disulfide-bound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species . Cyclotide-containing fractions of a peptide library revealed potent bioactivities against the Gram-negative bacteria ATCC 25922 and the highly virulent and multidrug-resistant VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 μM against and , respectively, whereas isolated cyI3 killed ∼50% of at 60 μM and isolated cyI4 demonstrated no killing at concentrations >60 μM against both pathogens. This work expands the repertoire of bioactive cyclotides found in . and highlights the potential of these antibacterial cyclic peptides.