Biofilm formation on biomedical implant surfaces requires bacterial adhesion, which increases the risk ofinfection and chronic inflammation. Since intercalation of quaternary ammonium salts (QAS) into montmorillonite(MMT) clay, known as organoclays, has been reported to increase surface broad-spectrumantibacterial properties, we aimed to develop an antibacterial surface composed of thermoplastic polyurethane(TPU) embedded with bentonite and MMT clay containing QAS to prevent initial bacterialattachment. We evaluated its potential application in reducing bacterial adhesion and enhancingbacteria-killing properties using Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results demonstrated that the nanoclay-embedded TPU surfaces with QAS significantly reduced theadhesion of E. coli and S. aureus by 68.82% and 65.18%, respectively, compared to the plain TPU surfaces. Additionally, a higher nanoclay concentration coating on the surface could enhance its effectiveness, asshown by 85.34% and 82.74% reduction in E. coli and S. aureus adhesion and killing efficiency. Furthermore, we observed that nanoclay-embedded TPU surfaces had no detrimental effects on the viabilityof human dermal fibroblasts. Taken together, these techniques could provide novel strategies forinhibiting bacterial adhesion and supporting bacteria killing on biomedical implant surfaces, as the investigatedsurfaces are simple to synthesize, efficient, and cost-effective.