Antibacterial resistance poses a critical public health threat, challenging the prevention and treatment of bacterial infections. The search for innovative antibacterial agents has spurred significant interest in quaternary heteronium salts (QHSs), such as quaternary ammonium and phosphonium compounds as potential candidates. In this study, a library of 49 structurally related QHSs was synthesized, varying the cation type and alkyl chain length. Their antibacterial activities against , including antibiotic-resistant strains, were evaluated by determining minimum inhibitory/bactericidal concentrations (MIC/MBC) ≤ 64 µg/mL. Structure-activity relationship analyses highlighted alkyl-triphenylphosphonium and alkyl-methylimidazolium salts as the most effective against CECT 976. The length of the alkyl side chain significantly influenced the antibacterial activity, with optimal chain lengths observed between C and C . Dose-response relationships were assessed for selected QHSs, showing dose-dependent antibacterial activity following a non-linear pattern. Survival curves indicated effective eradication of CECT 976 by QHSs at low concentrations, particularly compounds , , and . Moreover, in vitro human cellular data indicated that compounds , , and showed favourable safety profiles at concentrations ≤ 2 µg/mL. These findings highlight the potential of these QHSs as effective agents against susceptible and resistant bacterial strains, providing valuable insights for the rational design of bioactive QHSs.