In this study, fibre laser nitriding in open air was applied to the Ti6Al4V alloy in order to improve the wear resistance, thus minimising the generation of wear debris from the surfaces for load-bearing applications. The recent technological advancement to perform the laser nitriding process in open air allows the opportunity to surface-harden any curved and/or specific areas in the hip implants. The laser nitriding process was modulated between the pulsed mode and continuous wave (CW) mode by varying the duty cycle between 60% (pulsed) and 100% (CW). Our experimental investigations were divided into two stages in sequential order: Firstly, to create crack-free, homogenous and golden laser-nitrided surfaces by the proper selection of duty cycle. Secondly, it was to analyse the properties (both physical and chemical) of the wear debris as well as to evaluate their cytotoxicity and antibacterial performance. The laser-nitrided surfaces were characterized and tested using a variety of techniques, incl. Optical microscopy, SEM-EDX, XRD, surface roughness and Vickers hardness measurements, as well as tribological tests (i.e. ball-on-disk wear tests and DLS). The wear debris from the laser-nitrided surfaces (collected in the wear tests) were analysed using TEM, XPS and SEM-EDX. Their toxicity was evaluated using in-vitro cell culture with macrophages at two time points (24 h and 48 h). The antibacterial performance was tested in vitro against two of the most commonly implicated pathogens in orthopaedic infection, namely Staphylococcus aureus and Escherichia coli for 24 h. Our findings indicated that the wear resistance of the surfaces after laser nitriding was significantly improved and the amount of wear debris generated was also significantly reduced. The wear particles from the laser-nitrided surfaces were in the nano-sized scale range (0.01 μm to 0.04 μm or 10 nm to 40 nm). They were found to be less toxic towards RAW 264.7 macrophages, yet display antimicrobial properties against Staphylococcus aureus, when compared with the larger particles (1.5 μm in size) from the untreated surfaces. It is envisioned that successful fabrication of the non-toxic and highly wear-resistant TiN layer in Ti6Al4V using the open-air laser nitriding technique can enable progress towards the development of metal-on-metal (MoM) hip implants fully made of Ti-based alloys.