<p>Bacterial infections are becoming the second most common cause of death globally and have contributed significantly to morbidity and mortality. Cationic antibacterial polymers containing quaternary ammonium salts have been explored; however, it remains a key scientific challenge for current research to synergistically optimize the conformational relationships between structural surface features, active sites, and properties. In this study, a novel cationic copolymer microsphere with nano-multiple humps (CPMs-nMHs) was constructed through emulsion polymerization and self-assembly in EtOH/H₂O, with 3-methacrylamido-<i>N</i>,<i>N</i>,<i>N</i>-trimethylpropan-1-aminium chloride (MPAC) as the protruding functional component. Meanwhile, different hydrophilic monomers were adjusted to synthesize polymers with varying forms, which offered a significant research foundation for delving deeper into the impact of their morphology on performance. After being characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and thermogravimetric analysis (TG), the obtained CPMs-nMHs were applied to antibacterial activity against <i>Escherichia coli</i> (<i>E. coli</i>) and <i>Staphylococcus aureus</i> (<i>S. aureus</i>). Surprisingly, the synthesized CPMs-nMHs exhibited excellent antibacterial performance, discovering that the antibacterial rates of up to 100%, while the activities of contrast copolymers were low. We considered that the dual cooperation of cationic structures and nano-multiple humps were responsible for the antibacterial capabilities. Taken together, cationic copolymer microspheres with nano-multiple humps provide a promising strategy for enhancing the antibacterial properties of cationic polymers.</p>