Multifunctional polyurethane based on polysiloxane tethered cationic biocides was designed for fouling resistance and antibacterial objectives. Quaternary ammonium salt (QAS) functionalized hydroxy-terminated polysiloxanes (PQMS) with different compositions were synthesized by hydrolytic polycondensation and subsequent quaternization. Then, a series of antibacterial polyurethane copolymers (PQMS–PU) with PQMS and PPG as soft segments were prepared by two-step condensation methods. GPC, FTIR and ¹H-NMR were employed to analyze the chemical structural and composition. The surface migrations of polysiloxane segment tethered QAS groups and the distribution of N⁺ on the surface were quantitatively characterized by XPS. The microphase morphology and wettability properties of the copolymer films were investigated by AFM, TEM and dynamic contact angle tests. The migration of the biocide groups to the surface caused by the microphase separation between polysiloxane soft segments and hard segments was determined. The quantitative relationships of the polysiloxane segments and QAS group distribution on the surface with the wettability and antibacterial activity of the copolymer films were demonstrated. Within the designed range, the obtained copolymers exhibited very low surface free energy and excellent antibacterial activity towards Escherichia coli, which was of great environmental friendliness and industrial interest for textile and marine coating application.