In response to the urgent need for new antibiotic development strategies, antimicrobial peptides (AMPs) and other synthetic polymers are being actively investigated as promising alternatives to traditional antibiotics. Although most AMPs display lytic activity against several types of bacteria, they have poor toxicology profiles and are susceptible to proteolysis in vivo. While many synthetic variants have been created to mimic AMPs by tuning the hydrophobic to cationic ratio of the side-chain groups, few have decoupled the effects of charge from hydrophobicity in discrete systems, and none have investigated the effect of backbone hydrophobicity. We recently developed a rapid and efficient approach for the assembly of synthetic sequence-defined oligothioetheramides (oligoTEAs) that are resistant to protease activity. Our oligoTEA assembly scheme allows direct access to the oligomer backbone, which enables precise tuning of oligoTEA hydrophobicity while keeping charge constant. In this study, we synthesized a new class of antibacterial oligoTEAs (AOTs) with precise control over backbone hydrophobicity and composition. Our studies suggest that AOTs lyse cells via membrane permeabilization and that hydrophobicity and macromolecular conformation are key properties that regulate AOT activity. Some of our AOTs show highly promising antibacterial activity (MIC 0.5-5 μM) against clinically relevant pathogens in the presence of serum, with little to no toxicity against RBCs and HEK293 cells. Taken together, our data identify design parameters and criteria that may be useful for assembling the next generation of potent and selective AOTs. © 2017 American Chemical Society.