Photodynamic therapy is a promising approach for fighting bacterial infections because it can induce few side effects, develop no drug resistance, and realize precise treatment. However, most photosensitizers (PSs) have the disadvantages of poor water-solubility, severe self-quenching, and potential toxicity. Here, the cationic polymer polyethyleneimine (PEI) was used to prepare a cholesterol- and chlorin e6 (Ce6, a common PS)c-onjugated compound via the carboxyl-amine reaction or the acyl chloride-amine reaction (abbreviated as Chol-PEI-Ce6). The as-prepared Chol-PEI-Ce6 molecules can self-assemble into close-to-spherical nanoparticles (NPs) with an average diameter of similar to 15 nm and can bind to the bacterial surfaces via the synergistic hydrophobic insertion of the cholesterol moieties and electrostatic interaction between the cationic amine groups of PEI and the bacterial surfaces. Upon light irradiation, the NPs can effectively inactivate both Gram-positive and Gram-negative bacteria. Besides, the interaction between Chol-PEI-Ce6 NPs and bacteria markedly enhances the production of intracellular reactive oxygen species after light irradiation, which may account for the excellent antibacterial performance of the NPs. More importantly, the NPs possess negligible dark cytotoxicity and good hemocompatibility. Therefore, the present work may have strong implications for developing novel antibacterial agents to fight against bacterial infections.