Biophysical analysis into the mechanism of action of membrane-disrupting antibiotics such as antimicrobial peptides (AMPs) and AMP mimetics is necessary to improve our understanding of this promising but relatively untapped class of antibiotics. We evaluate the impact of cationic nature, specifically the presence of guanidine versus amine functional groups using sequence-defined oligothioetheramides (oligoTEAs). Relative to amines, guanidine groups demonstrated improved antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). To understand the mechanism of action, we evaluated membrane interactions by performing a propidium iodide assay and fluorescence microscopy of supported MRSA mimetic bilayers treated with oligoTEAs. Both studies demonstrated membrane disruption, while fluorescence microscopy showed the formation of lipid aggregates. We further analyzed the mechanism using surface plasmon resonance with a recently developed two-state binding model with loss. Our biophysical analysis points to the importance of lipid aggregation for antibacterial activity and suggests that guanidine groups improve antibacterial activity by increasing the extent of lipid aggregation. Altogether, these results verify and rationalize the importance of guanidines for enhanced antibacterial activity of oligoTEAs, and present biophysical phenomena for the design and analysis of additional membrane-active antibiotics.