Preparation of antibacterial polyurethane coatings from novel functional soybean oil was considered in this work. First, epoxidized soybean oil (ESBO) as a low price and widely available renewable resource raw material was subjected to the reaction with aniline using an ionic liquid as a green catalyst. The intermediate phenylamine containing polyol (SAP) was then methylated by reaction with methyl iodide to produce a polyol (QAP) with pendant dimethylphenylammonium iodide groups. To regulate the physical and mechanical properties as well as biological characteristics of final coatings, QAP was mixed with different portions of a similar soybean oil-based polyol (MSP) without quaternary ammonium groups. The mixtures were reacted with isophorone diisocyanate to produce crosslinked polyurethane coatings. Evaluation of viscoelastic properties by DMA method revealed single phase structure with Tg in the range of 50–82°C. Stress–strain analysis of the prepared polyurethanes showed initial modulus, tensile strength, and elongation at break in the ranges of 13–299 MPa, 4.5–13.8 MPa, and 16–109%, respectively. Additionally, the coatings showed good adherence to aluminum and PVC substrates. The solvent extracted samples showed excellent biocompatibility as determined by monitoring L929 fibroblast cells morphology and MTT assay. Meanwhile, very promising antibacterial properties against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria with bacterial reduction in the range of 83–100% was observed. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.