Stacking fault energies (SFE) were determined in additively manufactured (AM) stainless steel (SS 316 L) and equiatomic CrCoNi medium-entropy alloys. AM specimens were fabricated via directed energy deposition and tensile loaded at room temperature. In situ neutron diffraction was performed to obtain a number of faulting-embedded diffraction peaks simultaneously from a set of (hkl) grains during deformation. The peak profiles diffracted from imperfect crystal structures were analyzed to correlate stacking fault probabilities and mean-square lattice strains to the SFE. The result shows that averaged SFEs are 32.8 mJ/m2 for the AM SS 316 L and 15.1 mJ/m2 for the AM CrCoNi alloys. Meanwhile, during deformation, the SFE varies from 46 to 21 mJ/m2 (AM SS 316 L) and 24 to 11 mJ/m2 (AM CrCoNi) from initial to stabilized stages, respectively. The transient SFEs are attributed to the deformation activity changes from dislocation slip to twinning as straining. The twinning deformation substructure and atomic stacking faults were confirmed by electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). The significant variance of the SFE suggests the critical twinning stress as 830 ± 25 MPa for the AM SS 316 L and 790 ± 40 MPa for AM CrCoNi, respectively.