This research investigates the effect of the formation of an oxygen-stabilised titanium alpha layer – called alpha-case at the surface – on the fatigue properties of Ti-6Al-4V (Ti64) alloy components produced by Laser Powder Bed Fusion (L-PBF). Three post processing heat treatments with different controlled atmospheres were carried out on samples with as-built surfaces to evaluate how differences in alpha-case layer thickness and hardness affect the material’s susceptibility to surface embrittlement and its overall fatigue performance. The investigation includes bulk and subsurface microstructural analysis, surface characterisation by X-ray computed tomography (XCT), and fatigue testing. Key findings show that alpha-case layers can reduce the fatigue resistance of L-PBF fabricated Ti64. The presence of a 70±3 µm thick alpha-case layer was found to promote crack initiation. This is emphasised by a higher density of initiated cracks, thus leading to a reduction in fatigue life. Conversely, thinner alpha-case layers were found to have a reduced impact on the fatigue performance, highlighting the critical role of post processing heat treatments in modulating the fatigue resistance of the material. The use of XCT to characterise the surfaces of the specimens in 3D confirms that fatigue cracks primarily initiate at surface notches, highlighting the predominance of as-built surfaces over microstructure in determining the fatigue resistance of L-PBF Ti64 components.