Ti alloys are widely used in several fields, such as aerospace and biomedical, due to their high mechanical properties under severe loading conditions. Recently, the interest in these materials produced by additive manufacturing process has increased, but intensive research should be done to better characterise their properties. This work aims to study and compare the effect of surface properties, internal defects, microstructure, hardness and Hot Isostatic Pressing (HIP) treatment or in-Vacuum Heat Treatment (VHT) on fatigue properties of a Ti6Al4V produced by Selective Laser Melting (SLM) and Electron Beam Melting (EBM) additive manufacturing technologies. The samples were fully characterised using a wide range of techniques, in terms of microstructure (optical microscopy and SEM), mechanical properties (hardness mapping) and surface texture (confocal microscopy). The internal defects were evaluated using an image-based analysis approach. The uniaxial fatigue endurance limit properties were determined by a Dixon-Mood staircase approach and the failed samples near the fatigue limit were characterised by fracture surface and defect area analysis. A study of the applied load on the flaw areas was carried out to unveil the root causes of fatigue failure. The results showed that the fatigue properties of the as-printed samples were mainly determined by the surface roughness, whereas in the machined samples the internal defect dimension ruled the fatigue resistance of the material. The HIP used as a post-printing treatment is effective in substantially reducing the presence of internal pores, although local microstructural changes can take place only in the case of smooth surface. In conclusion, when properly developed in their melted parameters, both EBM and SLM technologies produce similar mechanical performance on comparable roughness levels, thus finding shared fields of application and fully eligible for the production of structural components.