The effect of crystal orientation and twinning on the mechanical characteristics and microstructure development of the equiatomic FeNiCrCoCu high-entropy alloy is explored and quantified by nanoindentation using molecular dynamics. The substrate hardness and discontinuity in the loading curves are strongly influenced by various crystal orientations due to differences in microstructural evolution and dissimilar dislocation events. The Hall-Petch aspect representing the correlation between the twinning distance and the material strength is found with decreasing the twin boundary distance. The plastic deformation exposes that the propagation of local stress and shear strain into the substrate bulk is dependent on crystal orientation, characterized through the various directions of the slipping bands. Meanwhile, the growth of shear bands into the substrate is easier with the great twin boundary distance, which can enhance the ductility of the alloy. The microstructural evolution suggests that the sliding prismatic dislocation loops in the substrate play a significant role in deformation. The displacement of the dislocation loops into the substrate depends on the crystal orientation, and their propagation is inhibited by the twin boundary. The various surface morphologies of the substrate are dominated by the sliding mechanism of the atoms following the symmetry of< 1 1‾ 0>{111} primary sliding system. Besides, the pile-up height tends to increase with the decrease of the twinning distance because the interaction between the sliding system and the twinning occurs more for the small twin boundary distance.