This letter is aimed to develop a general strategy to fabricate polypseudorotaxanes with controlled antibacterial activity based on cationic polymers. As a proof of concept, the commercially available antibacterial cationic polymer, ε-poly-l-lysine hydrochloride, was chosen for the demonstration. Using host–guest chemistry, cucurbituril (CB), a water-soluble macrocyclic host, was employed to bind with the positive charge and hydrophobic component on ε-poly-l-lysine hydrochlorides for antibacterial regulation. In this way, by tuning the ratio of CB to the cationic polymer, the antibacterial polypseudorotaxane can be obtained, and the antibacterial efficiency can be well tuned from 5% to 100%. This line of research will enrich the field of cationic polymers and polypseudorotaxanes with important functions on precise control over antibacterial activity.