We prepared a class of novel cationic polymers as antimicrobial agents: quaternary ammonium-containing poly(N,N-dimethylaminoethyl methacrylate) with natural rosin as the pendant group (PDMAEMA-g-rosin). Different from most other amphipathic antimicrobial polymeric systems reported in the literature, our approach sandwiched the hydrophilic cationic group between the polymer backbone and bulky hydrophobic hydrophenanthrene side groups. A simple quaternization reaction was used to link the rosin ester chloride and PDMAEMA homopolymers. Both the Gram-positive bacterium Staphylococcus aureus (S. aureus) and Gram-negative bacterium Escherichia coli (E. coli) were tested against the PDMAEMA-g-rosin copolymers. PDMAEMA-g-rosin copolymers with the amphipathic structure exhibited effective antimicrobial activity against both E. coli and S. aureus. Both the degree of quaternization of rosin group and the molecular weight of PDMAEMA played roles in antimicrobial activities. Our results also indicated that conformation of hydrophobic group (particularly steric hindrance) played a role in dictating antibacterial efficacy. Scanning electron microscopy and confocal laser scanning microscopy were used to characterize morphological changes of bacteria after exposure with PDMAEMA-g-rosin copolymers. Possible mechanisms on a combination of ionic and hydrophobic interactions between bacterial cells and polymers are discussed.