Polymer microspheres having bio-based cardanol moiety were prepared via suspension polymerization of 2-hydroxy-3-cardanylpropyl methacrylate (CM). The incorporation of the cardanol moiety into polymer microspheres could impart the bactericidal properties and the microspheres were found to have a cross-linked structure formed during the suspension polymerization by the reactions between the double bonds in the cardanol moieties. The cross-linked structure is very useful to prepared physically stable polymer films using the polymer microsphere. The polymer microsphere itself prepared by CM and the polymer film prepared by casting the polymer microsphere suspension were found to have very high antibacterial activity against E.coli; the minimum inhibition concentration (MIC) value of the polymer microsphere against E.coli was 0.403 mg mL−1 and the antibacterial activity of the polymeric film prepared by casting the polymer microsphere suspension against E.coli was larger than 99.9%. Copolymer microspheres prepared by the suspension copolymerization of CM with styrene(ST) and the polymer films prepared from the copolymer microspheres also showed high antibacterial activity. For example, the MIC value of the copolymer microsphere prepared using 80 to 20 mol ratios of ST to CM was 0.519 mg mL−1 and the antibacterial activity of the polymer films of the copolymer microsphere is 99.9%. The bactericidal properties of the polymer films were found to be caused by the disruption of cell wall by contact-killing without any leaching of antibacterial moieties. Furthermore, all the polymeric films from microsphere of the polymer and copolymers showed sufficient biocompatibility, regardless of their monomer compositions.