The one-pot synthesis of antimicrobial bottle brush copolymers is presented. Reversible addition-fragmentation chain-transfer (RAFT) polymerization is used for the production of the polymeric backbone, as well as for the grafts, which were installed using a grafting-from approach. A combination of N-isopropyl acrylamide and a Boc-protected primary amine-containing acrylamide was used in different compositions. After deprotection, polymers featuring different charge densities were obtained in both linear and bottle brush topologies. Antimicrobial activity was tested against three clinically relevant bacterial strains, and growth inhibition was significantly increased for bottle brush copolymers. Blood compatibility investigations revealed strong hemagglutination for linear copolymers and pronounced hemolysis for bottle brush copolymers. However, one bottle brush copolymer with a 50% charge density revealed strong antibacterial activity and negligible in vitro blood toxicity (regarding hemolysis and hemagglutination tests) resulting in selectivity values as high as 320. Membrane models were used to probe the mechanism of shown polymers that was found to be based on membrane disruption. The trends from bioassays are accurately reflected in model systems indicating that differences in lipid composition might be responsible for selectivity. However, bottle brush copolymers were found to possess increased cytotoxicity against human embryonic kidney (HEK) cells compared with linear analogues. The introduced synthetic platform enables screening of further, previously inaccessible parameters associated with the bottle brush topology, paving the way to further improve their activity profiles.