A substrate with a multilayer reflective film capable of facilitating the discovery of contaminants, scratches and other critical defects by inhibiting the detection of pseudo defects attributable to surface roughness of a substrate or film in a defect inspection using a highly sensitive defect inspection apparatus.The substrate with a multilayer reflective film has a multilayer reflective film obtained by alternately laminating a high refractive index layer and a low refractive index layer on a main surface of a mask blank substrate used in lithography, wherein an integrated value I of the power spectrum density (PSD) at a spatial frequency of 1 μm−1 to 10 μm−1 of the surface of the substrate with a multilayer reflective film, obtained by measuring a region measuring 3 μm×3 μm with an atomic force microscope, is not more than 180×10−3 nm3, and the maximum value of the power spectrum density (PSD) at a spatial frequency of 1 μm−1 to 10 μm−1 is not more than 50 nm4.