The one-pot synthesis of antimicrobial bottle brush copolymers is presented. RAFT polymerization is used for production of the polymeric backbone, as well as for the grafts, which were installed using a grafting from approach. A combination of N-iso propyl acrylamide and a Boc protected primary amine-containing acrylamide was used in different composition. After deprotection polymers featuring different charge densities were obtained in both, linear and bottle brush topology. Antimicrobial activity was tested against three clinically relevant bacteria strains and growth inhibition was significantly increased in 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 strong antibacterial activity and negligible blood toxicity resulting in selectivity values as high as 320. Membrane models were used to probe the mechanism of shown polymers, which was found to be based on membrane disruption. The trends from biology are accurately reflected in model systems indicating that differences in lipid composition are responsible for selectivity. However, bottle brush copolymers were found to possess increased cytotoxicity against HEK cells when compared with linear analogues. The introduced synthetic platform enables screening of further parameters associated to bottle brush copolymers, which might lead to even better activity profiles.