AbstractAntimicrobial‐resistant pathogens have reached alarming levels, becoming one of the most pressing global health issues. Hence, new treatments are necessary for the fight against antimicrobial resistance. Synthetic nanoengineered antimicrobial polymers (SNAPs) have emerged as a promising alternative to antimicrobial peptides, overcoming some of their limitations while keeping their key features. Herein, a library of amphiphilic oxazoline‐based SNAPs using cationic ring‐opening polymerization (CROP) is designed. Amphipathic compounds with 70% cationic content exhibit the highest activity against clinically relevant Staphylococcus aureus isolates, maintaining good biocompatibility in vitro and in vivo. The mechanism of action of the lead compounds against S. aureus is assessed using various microscopy techniques, indicating cell membrane disruption, while the cell wall remains unaffected. Furthermore, a potential interaction of the compounds with bacterial DNA is shown, with possible implications on bacterial division. Finally, one of the compounds exhibits high efficacy in vivo in an insect infection model.