Leishmaniasis, a category I neglected tropical disease, is a group of diseases caused by the protozoan parasite species with a wide range of clinical manifestations. Current treatment options can be highly toxic and expensive, with drug relapse and the emergence of resistance. Bacteriocins, antimicrobial peptides ribosomally produced by bacteria, are a relatively new avenue for potential antiprotozoal drugs. Particular interest has been focused on enterocin AS-48, with previously proven efficacy against protozoan species, including spp. Sequential characterization of enterocin AS-48 has illustrated that antibacterial bioactivity is preserved in linearized, truncated forms; however, minimal domains of AS-48 bacteriocins have not yet been explored against protozoans. Using rational design techniques to improve membrane penetration activity, we designed peptide libraries using the minimal bioactive domain of AS-48 homologs. Stepwise changes to the charge (), hydrophobicity (), and hydrophobic dipole moment (μ) were achieved through lysine and tryptophan substitutions and the inversion of residues within the helical wheel, respectively. A total of 480 synthetic peptide variants were assessed for antileishmanial activity against Leishmania donovani. One hundred seventy-two peptide variants exhibited 50% inhibitory concentration (IC) values below 20?μM against axenic amastigotes, with 60 peptide variants in the nanomolar range. Nine peptide variants exhibited potent activity against intracellular amastigotes with observed IC values of <4?μM and limited host cell toxicity, making them worthy of further drug development. Our work demonstrates that minimal bioactive domains of naturally existing bacteriocins can be synthetically engineered to increase membrane penetration against spp. with minimal host cytotoxicity, holding the promise of novel, potent antileishmanial therapies. Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus . There are three primary clinical forms, cutaneous, mucocutaneous, and visceral, with visceral leishmaniasis being fatal if left untreated. Current drug treatments are less than ideal, especially in resource-limited areas, due to the difficult administration and treatment regimens as well as the high cost and the emergence of drug resistance. Identifying potent antileishmanial agents is of the utmost importance. We utilized rational design techniques to synthesize enterocin AS-48 and AS-48-like bacteriocin-based peptides and screened these peptides against L. donovani using a fluorescence-based phenotypic assay. Our results suggest that bacteriocins, specifically these rationally designed AS-48-like peptides, are promising leads for further development as antileishmanial drugs.