This research program investigated the effects of layer thickness (50 µm and 100 µm) on the microstructure and mechanical properties of electron beam powder bed fusion (EBPBF) additive manufacturing of Inconel 625 alloy. The as-built 50 µm and 100 µm layer thickness components were also heat treated at temperatures above 1100 °C which produced a recrystallized grain structure containing annealing twins in the 50 µm layer thickness components, and a duplex grain structure consisting of islands of very small equiaxed grains dispersed in a recrystallized, large-grain structure containing annealing twins. The heat-treated components of the microstructures and mechanical properties were compared with the as-built components in both the build direction (vertical) and perpendicular (horizontal) to the build direction. Vickers microindentation hardness (HV) values for the vertical and horizontal geometries averaged 227 and 220 for the as-built 50 µm and 100 µm layer components, respectively, and 185 and 282 for the corresponding heat-treated components. The yield stress values were 387 MPa and 365 MPa for the as-built horizontal and vertical 50 µm layer geometries, and 330 MPa and 340 MPa for the as-built 100 µm layer components. For the heat-treated 50 µm components, the yield stress values were 340 and 321 MPa for the horizontal and vertical geometries, and 581 and 489 MPa for the 100 µm layer components, respectively. The elongation for the 100 µm layer as-built horizontal components was 28% in contrast with 65% for the corresponding 100 µm heat-treated layer components, an increase of 132% for the duplex grain structure.