Despite the rapid growth in cationic host defence peptide (CHDP)-mimicking synthetic antibacterial polymers, the importance of the well-defined secondary structure of synthetic polymers, similar to CHDP, has not been studied in the context of bacterial membrane perturbation, which is the key focus of this article. Amongst the seven tested amphiphilic cationic alternating copolymers, five (F-PUs) contain flexible (F) linear hydrocarbons (C4, C6, C8) and two (R-PUs) contain rigid (R) cyclic hydrocarbons (C6, C13) in the segmented biodegradable polyurethane (PU) backbone. F-PUs adopt an intra-chain H-bonding driven pleated structure, followed by hierarchical assembly, producing cationic polymersomes in water. In sharp contrast, R-PUs, deprived of such chain-folding possibility due to rigid linkers, exhibit immiscibility driven aggregation, producing spherical particles. F-PUs exhibit antibacterial activity with a rather low minimum inhibitory concentration (MIC), while R-PUs do not show even moderate activity. F-PUs with the C6 linker represent the most optimal balance between the hydrophobicity and the charge density and consequently exhibit the most lethal activity. F-PU-C6a with a pendant primary amine group shows exemplary activity selectively against Gram negative E. coli over Gram positive bacteria [MIC_{S. aureus}/MIC_{E. coli} = 64]. On the other hand, F-PU-C6b or F-PU-C6c with a pendant quaternary amine or guanidium group exhibits broad-spectrum activity with very low MIC values of ∼2.0 μg mL⁻¹ in some cases, which is rarely reported in the literature. While these polymers are lethal against bacteria, they exhibit negligible toxicity against mammalian cells as evident from the lack of hemolysis (HC₁₀ > 200 μg mL⁻¹) of red blood cells and MTT assay results. Mechanistic investigation establishes the cell-killing via a non-specific membrane disruption pathway, similar to CHDPs present in the innate immunity. Beyond planktonic bacteria, F-PUs also exhibit extraordinary biofilm eradication efficiency as ∼100% eradication of a 3-day aged biofilm is noticed with ∼30 μg mL⁻¹ F-PU-C6 in just 3 h. Antibacterial activity and biofilm eradication ability of selected polymers in the present series are comparable to the benchmark of polymyxin-B or levofloxacin, the well-known commercially available antibiotics.