Selective laser melting (SLM) is an additive manufacturing technique which permits fabrication of three dimensional parts by selectively melting consecutive layers of metallic powder. This allows the production of parts with high geometrical complexity. Titanium alloy Ti-6Al-4V (Ti64) is widely used in industry due to its high strength-to-mass ratio, corrosion resistance and biocompatibility. SLM increases the application range of Ti64 because of its flexibility for prototyping any part and its low material waste. Nitriding is a diffusion-based thermo-chemical treatment for interstitial hardening of the surface of Ti64 alloy products. This study characterized the fatigue behaviour of SLM-produced Ti64 nitrided and annealed bars in as-built and machined surface conditions. The surface of the SLM-produced Ti64 parts after gas nitriding showed high values of micro-hardness up to 550 HV just below the surface. Fatigue testing was performed to assess the materials fatigue strength and fractographic imaging was used to examine fracture surface and nitride layer characteristics. Nitriding was found to reduce the fatigue strength of the samples to a similar level irrespective of being in the as-built or machined condition. The effect of nitriding on crack initiation and growth at various stress levels under fatigue loading was investigated.