Fatigue crack initiation in the direct aged version of the nickel-based superalloy Inconel 718 has been investigated at room temperature in the low stress/very high cycle regime via ultrasonic fatigue testing. Three different microstructures have been examined at the same strain amplitude in order to understand the influence of non-metallic inclusions (NMIs), i.e. carbides, carbonitrides and nitrides, and Σ3 twin boundary density on lifetime and failure mode. A slight refinement in grain structure and a higher Σ3 twin boundary density is associated with substantial reductions in lifetime. Decreasing Σ3 twin boundary density for fine grain microstructures results in a change in crack initiation mechanism from strain localization within grains at the high end of the grain size distribution to cracking of NMIs. To study the early stages of crack initiation and growth, specimens with pre-cracked NMIs were also tested in order to examine the role of the surrounding grain structure. Pre-cracked NMIs mainly result in macroscopic failure initiation at NMIs independent of the wrought microstructure. However, pre-loading specimens within the plastic domain highlighted the competition in crack initiation mode between cracked-NMIs and favorably oriented twin boundaries. Crack arrest from most of the pre-cracked NMIs demonstrates that surrounding grain structure (grain orientation, local plasticity and roughness in the vicinity of crack tip due to pre-straining) play a key role in the fatigue life of components stressed in the nominal elastic regime.