AbstractCrystallization‐driven self‐assembly (CDSA) was employed for the preparation of monodisperse cationic cylindrical nanoparticles with controllable sizes, which were subsequently explored for their effect on antibacterial activity and the mechanical properties of nanocomposite hydrogels. Poly(ɛ‐caprolactone)‐block‐poly(methyl methacrylate)‐block‐poly[2‐(tert‐butylamino) ethyl methacrylate] (PCL‐b‐PMMA‐b‐PTA) triblock copolymers were synthesized using combined ring‐opening and RAFT polymerizations, and then self‐assembled into polycationic cylindrical micelles with controllable lengths by epitaxial growth. The polycationic cylinders exhibited intrinsic cell‐type‐dependent antibacterial capabilities against gram‐positive and gram‐negative bacteria under physiological conditions, without quaternization or loading of any additional antibiotics. Furthermore, when the cylinders were combined into anionic alginate hydrogel networks, the mechanical response of the hydrogel composite was tunable and enhanced up to 51%, suggesting that cationic polymer fibers with controlled lengths are promising mimics of the fibrous structures in natural extracellular matrix to support scaffolds. Overall, this polymer fiber/hydrogel nanocomposite shows potential as an injectable antibacterial biomaterial, with possible application in implant materials as bacteriostatic agents or bactericides against various infections.