An optimum hydrophilic/hydrophobic balance has been recognized as a crucial parameter in designing cationic polymers that mimic antimicrobial peptides (AMPs). To date, this balance was achieved either by hydrophilicity variation through altering the nature and the number of cationic charges or by hydrophobicity modulation through incorporation of alkyl groups of different chain lengths. However, how the hydrophobicity variation through AMPs' building blocks-amino acids-influences the antibacterial efficacy of AMP-mimicking cationic polymers has rarely been explored. Toward this goal, herein we report a class of amino acid conjugated polymers (ACPs) with tunable antibacterial activity through a simple post-polymer-functionalization strategy. Our polymeric design comprised a permanent cationic charge in every repeating unit, whereby the hydrophobicity was tuned through incorporation of different amino acids. Our results revealed that the amino acid alteration has a strong influence on antibacterial efficacy. Upon increasing the amino acid side-chain hydrophobicity, both the antibacterial activity (against broad spectrum of bacteria) and toxicity increased. However, the distinct feature of this class of polymers was their good activity against -the top most critical pathogen according to WHO, which has created an alarming situation worldwide, causing the majority of infections in humans. A nontoxic (no hemolysis even at 1000 μg/mL) ACP including a glycine residue (ACP- (Gly)) showed very good activity (MIC = 8-16 μg/mL) against both drug-sensitive and drug-resistant strains of , including clinical isolates. This polymer not only was rapidly bactericidal against growing planktonic but also killed nondividing stationary-phase cells instantaneously (<2 min). Moreover, it eradicated the established biofilm formed by drug-resistant clinical isolates. No propensity for bacterial resistance development against this polymer was seen even after 14 continuous passages. Taken together, the results highlight that hydrophobicity modulation through incorporation of amino acids in cationic polymers will provide a significant opportunity in designing new ACPs with potent antibacterial activity and minimum toxicity toward mammalian cells. More importantly, the excellent anti- efficacy of the optimized lead polymer indicates its immense potential for being developed as therapeutic agent.