The aim of this study is to evaluate the effect of two nitriding heat treatments on high cycle fatigue of Ti64 (Grade 5) alloy. For this, axial fatigue tests have been performed on untreated and nitrided round bar specimens under constant amplitude load-controlled mode at a frequency of 20 Hz at room temperature and in laboratory air. After the testing, the fractured surfaces and microstructures were investigated by scanning electron microscopy, focused ion beam, transmission electron microscopy and scanning transmission electron microscopy. Cross-sectional studies show that two different layers were formed after the nitriding processes, namely, a top compound layer and a diffusion layer below it. Additionally, a very thin layer rich in aluminium was also detected. The fatigue results show that the nitriding treatments are detrimental to the fatigue life when compared to the untreated and extruded Ti64 alloy. The main conclusion is that the reduction of high cycle fatigue life is a result of the cracking of the nitrided layers.