In this work, the single-layered amorphous NbMoTaW and multilayered amorphous NbMoTaW/TiVCr high entropy alloys were prepared to investigate the diffusion barrier performance. The microstructure and thermal stability of these samples were analyzed by X-ray diffraction, transmission electron microscope, and four-point probe method after annealing at different temperatures. In the single-layered structure, Cu silicide formed after annealing at 600 °C. The reason is that the amorphous NbMoTaW layer crystallizes during annealing, which causes the interdiffusion between Cu and Si through grain boundaries (GBs). However, the multilayered structure still maintains effective barrier performance as the annealing temperature is below 800 °C. Amorphous structure without GBs diffusion path, severe lattice distortion, and the high dense layer interface are responsible for the improved barrier property in the multilayered system.