Owing to their attractive structure and mechanical properties, high-entropy alloys (HEAs) and medium-entropy alloys (MEAs) have attracted considerable research interest. The strength of HEAs/MEAs with a single face-centered cubic (FCC) phase, on the other hand, requires improvement. Therefore, in this study, we demonstrate a strategy for increasing the room-temperature strength of FCC-phase HEAs/MEAs by tuning cryo-pre-straining-induced crystal defects via the temperature-dependent stacking fault energy-regulated plasticity mechanism. Through neutron diffraction line profile analysis and electron microscope observation, the effect of the tuned defects on the tensile strength was clarified. Due to the cryo-rolling-induced high dislocation density, mechanical twins, and stacking faults, the room-temperature yield strength of an equiatomic CoCrFeNi HEA was increased by ∼290%, from 243 MPa (as-recrystallized) to 941.6 MPa (30% cryo-rolled), while maintaining a tensile elongation of 18%. After partial recovery via heat treatment, the yield strength and ultimate tensile strength decreased slightly to 869 and 936 MPa, respectively. Conversely, the elongation increased to 25.6%. The dislocation density and distribution of the dislocations were found to contribute to the strengthening caused by forest dislocations, which warrants further investigation. This study discussed the possibility of developing single-phase high-performance HEAs by tuning pre-straining-induced crystal defects.