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Many reported polycations show high antimicrobial activity along with low hemolytic activity. Unfortunately, most of those molecules remain highly cytotoxic against various mammalian cells. In this work, a systematic study on the impact of triethylene glycol monomethyl ether side groups (short polyethylene glycol (PEG) analog) on antimicrobial, hemolytic, and cytotoxic properties of novel amphiphilic ionenes is presented. A detailed description of synthesis, leading to well\u2010defined alternating polymers, which differ in structural elements responsible for hydrophilicity (PEG) and hydrophobicity (alkyl chain), is presented. Obtained results show that the PEG moiety and fine\u2010tuned hydrophilic\u2010lipophilic balance of ionenes synergistically lead to low cytotoxic, low hemolytic molecules with high activity against <jats:italic>S. aureus<\/jats:italic>, including methicillin\u2010resistant strains (MRSA). 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