Nature ubiquitously utilizes precise chemical arrangement to create and tune advanced structures and interactions. Within synthetic and natural scaffolds, this arrangement can often be read as a “sequence,” which creates particular functions and/or phenotypes. Much research has sought to understand chemical sequences to guide the design of molecules and materials for therapeutic benefit. Examples and additional background are discussed in Chapter 1. In this work, I have explored a new class of sequence-defined polymers called oligothioetheramides (oligoTEAs) described in Chapter 2 in search of sequence-structure-function relationships by employing solution-phase and biophysical characterization. In Chapter 3, we demonstrate the challenging solution-phase structural characterization of a flexible sulfonated oligoTEA, utilizing a range of techniques including variable temperature pulse field gradient (PFG) NMR, double electron−electron resonance (DEER), molecular dynamics (MD) simulations, as well as the combination of PFG NMR and DEER within Stokes−Einstein−Sutherland diffusion theory. I apply these techniques along with biophysical characterization toward a specific class of antibacterial oligoTEAs to evaluate their potential as antibiotics. Motivated by the need to new antibiotic development, these oligoTEAs are designed as membrane-targeting AMP mimetics, featuring cationic and hydrophobic groups to potently and selectively disrupt bacterial membranes. However, this physical design has struggled to guide broad success for AMPs in vivo. Thus, we have investigated additional properties for optimization of these antimicrobials. We completed solution-phase characterization including small- and wide-angle x-ray scattering (SAXS/WAXS), as well as fluorescence microscopy and surface plasmon resonance (SPR), both with Staphylococcus aureus mimetic membranes. In Chapter 4, I have examined the characterization a pair of constitutional oligoTEA isomers that show a unique ~ 10-fold difference in antibacterial potency. In Chapter 5, I present the characterization of oligoTEAs with different cationic groups and with hydrophobic backbone sequences. Across all studies, oligoTEAs direct the formation of multimeric lipid aggregates that correlates with biological activity and helps establish a framework for the kinetic mechanism of action. Thus, revealing new parameters for antimicrobial optimization. Overall, this work highlights the importance of sequence definition and biophysical characterization for the design of new membrane-targeting antibiotics.