In response to the increasing threat of drug-resistant bacteria, numerous antibacterial peptide (AMP) mimics have been developed and shown substantial efficacy in killing bacteria. However, controlling the secondary structure of these mimics and clarifying the relationship between their conformation and antibacterial activity remain significant challenges. This study integrates facially amphiphilic bile acid scaffolds with chiral lysine centers to construct a series of bile acid–lysine polymers (BALP-n) as AMP mimics. Incorporating chiral centers into the polymer backbone allowed the induction and modulation of a stable antiparallel β-sheet secondary structure, reinforced by hydrogen bonding and hydrophobic interactions. For example, BALP-1 exhibited 51.9% antiparallel β-sheet content at pH 7.4 and demonstrated rapid bactericidal activity against Gram-positive bacteria, Gram-negative bacteria, and drug-resistant strains. In a weakly acidic environment, the antiparallel β-sheet content increased to 56.2%, accompanied by enhanced antibacterial performance. This chirality-driven strategy enables pH-responsive “on-demand” activation and improved membrane selectivity, distinguishing it from conventional cationic antibacterial polymers. BALP-1 also showed high stability under elevated temperatures and in the presence of proteinase K. Moreover, these AMP mimics displayed excellent biocompatibility, potent in vivo antibacterial activity, and the ability to promote wound healing, offering a promising approach to combat drug-resistant bacteria and biofilms.