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Premature implant failure, a critical concern in biomedical applications, is often attributed to poor biocompatibility and vulnerability to bacterial colonization. These issues are addressed by creating an endoprosthetic material with natural biocompatibility and antibacterial properties. In this in vitro study, the relaxed and unrelaxed tetrahedral amorphous carbon (ta-C) coatings were examined, both fabricated by the improved patented Pulsed Laser Deposition (PLD) technology. The chemical composition, surface roughness, hardness, topography, and wettability were analyzed. The ta-C surfaces were incubated by MM6 cells, E. coli and S. capitis bacteria for 24 h. PCR assessed the inflammatory response in MM6 cells, while fluorescence microscopy quantified adhering bacteria, and scanning electron microscopy examined local adhesion behavior. The results demonstrate comparable carbon phase composition, wettability properties, and hardness for both relaxed and unrelaxed ta-C. However, relaxed ta-C coating exhibited significantly fewer defects in terms of both quantity and quality, along with an antibacterial effect against E. coli. This suggests that the relaxed ta-C coating could contribute to the development of an endoprosthesis, preventing adverse biological reactions and implant-related infections, thus improving the longevity of the prosthesis., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)