Creating hierarchical polymer brushes possessing antifouling and bactericidal functionalities is a promising approach to combat biomaterial-associated infections. Hence, a well-constructed hierarchical structure is required to achieve optimized antibacterial performance. In this work, contact-killing cationic bactericidal poly(quaternary ammonium salts) (PQAs) bearing different alkyl chain lengths and zwitterionic antifouling poly(sulfobetaine methacrylate) (PSBMA) functional segments were grafted onto an activated substrate via surface-initiated atom transfer radical polymerization (SI-ATRP), and three kinds of polymer brushes with different architectures (Si-PQAs-b-PSBMA, Si-PSBMA-b-PQAs and Si-PQAs-r-PSBMA) were constructed. We demonstrate that the antibacterial effect simultaneously depends on the alkyl chain lengths of PQAs and the hierarchical structure of cationic/zwitterionic segments in polymer brushes. When the polymer brushes composed of a bactericidal bottom layer and an antifouling top layer, the ideal alkyl chain length of PQAs should be eight carbon atoms (Si-PQA8C-b-PSBMA), while in the opposite hierarchical structure, the optimized alkyl chain length of PQAs to synergize with PSBMA was four carbon atoms (Si-PSBMA-b-PQA4C). By appropriately adjusting the alkyl chain length or the hierarchical architecture, the interference between the antifouling and bactericidal functions could be avoided, thus achieving the outstanding long-term antibacterial performance against S. aureus, as well as good hemocompatibility and low cytotoxicity. This work provides fundamental guidance for the design and optimization of efficient and reliable antibacterial surfaces to inhibit biofilm formation.