Antimicrobial polymers as mimics of natural antimicrobial peptides are emerging as an alternative to classic antibiotics due to their potency, selectivity and lower susceptibility to resistance. The key chemical aspects necessary to confer high activity and selectivity to the polymer chain composition are largely known. However, little attention has been paid to how end-groups affect the overall biological activity. Here we report the use of RAFT polymerization to obtain eight well-defined cationic methacrylate polymers which bear either amine (PA1-4) or guanidine (PG1-4) pendant groups, while systematically varying the R- and Z-RAFT end-groups. These polymers were assessed in haemotoxicity assays as well as antimicrobial testing against clinically relevant pathogens; such as a vigorously biofilm forming strain of Staphylococcus epidermidis (S. epidermidis) and a vancomycin and methicillin resistant strain of Staphylococcus aureus (VISA) as well as the opportunistic fungus Candida albicans (C. albicans). The R-group was found to dominate the measured toxicity of polymers. Replacement of the anionic cyanovaleric acid R-group (PA1) with the neutral isobutyronitrile (PA3) led to over a 20 fold increase in the haemolytic activity of the polymers. The Z-group, however, was found to have more influence on the antimicrobial activity of the polymers against both VISA and C. albicans, whereby polymers with a long, lipophilic dodecylsulfanyl Z-group (PA1) were found to be more potent than those with either an ethylsulfanyl or no ZCS2-group. These results indicate that chemical control over the end-groups is a key element for achieving the desired high biological activity and selectivity, particularly when low molecular weights are required for maximum antibacterial activity.