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 Viola spp. 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 Viola inconspicua. Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gram-negative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae 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 E. coli and K. pneumoniae, respectively, whereas isolated cyI3 killed ∼50% of E. coli 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 Viola spp. and highlights the potential of these antibacterial cyclic peptides.