The effects of different surface pretreatment methods on the nucleation and growth of ultra-nanocrystalline diamond (UNCD) films grown from focused microwave Ar/CH4/H2 (argon-rich) plasma were systematically studied. The surface roughness, nucleation density, microstructure, and crystallinity of the obtained UNCD films were characterized by atomic force microscope (AFM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. The results indicate that the nucleation enhancement was found to be sensitive to the different surface pretreatment methods, and a higher initial nucleation density leads to highly smooth UNCD films. When the silicon substrate was pretreated by a two-step method, i.e., plasma treatment followed by ultrasonic vibration with diamond nanopowder, the grain size of the UNCD films was greatly decreased: about 7.5 nm can be achieved. In addition, the grain size of UNCD films depends on the substrate pretreatment methods and roughness, which indicates that the surface of substrate profile has a “genetic characteristic”.