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Titanium (Ti) and Ti alloys possess extensive applications in the medical field due to their non-toxic, biocompatible, and antibacterial properties. This study investigates the biocompatibility enhancement of a Ti-based alloy, Ti-6Al-4V, through a multi-step process involving cathodic arc ion plating of a TiZrTa thin film and subsequent laser-textured oxidation (LTO). LTO treatment produced a structured microtopography with regular groove patterns, including straight lines (L-s), circles (L-c), and checkboards (L-ch), resulting in surface oxidation and the formation of various oxides on the TiZrTa coating. Microstructure analyses revealed substantial alterations in surface topography induced by laser texturing. Antibacterial assessments against Staphylococcus aureus (S. aureus) showed that the films with LTO treatment did not exhibit significant advantages after 12 or 24 h of coculture because the laser-textured portions consisted of a combination of the film and the substrate, and the antibacterial effect was not obvious. Coatings with LTO treatment significantly improved L-929 mouse fibroblasts cell viability at 48 h, particularly for samples with a 50 μm spacing of straight and circular patterns (L-s and L-c). After two weeks of MG-63 human osteosarcoma cell culture, the viability of all samples reached similar levels. It suggested that the TiZrTa-coated Ti-6Al-4V exhibited good biocompatibility with both cells. This study contributed to the understanding of surface modification techniques for enhancing the biocompatibility of Ti-based alloys in biomedical applications. • A TiZrTa thin film with LTO treatment was applied onto Ti-6Al-4V alloys. • LTO produced regular groove patterns and resulted in superior hydrophilicity. • LTO-treated thin films did not have obvious antibacterial improvement of S. aureus. • LTO-treated thin films improved the cell viability of both L-929 and MG-63 cells. [ABSTRACT FROM AUTHOR]