The chemical reaction between Fe and lacquer has been used to create the black color in lacquer coatings since ancient times. Here, the effects of Fe ion addition, UV irradiation, and heating on the microscopic structures of black lacquer films were investigated by using X-ray absorption near edge structure (XANES), extended X-ray absorption fine structure (EXAFS), Fourier transform-infrared spectroscopy (FT-IR), small-angle X-ray scattering (SAXS), and small angle neutron scattering (SANS). The EXAFS result indicated that heating and UV irradiation made the coordination structure of Fe3+ in the lacquer nonuniform, and that heating caused the greatest nonuniformity. The FT-IR, SAXS, and SANS results demonstrated that the microscopic structural changes in the black lacquer films were induced by both heating and UV irradiation, but the changes were different. Heating caused a substantial structural change on the nanoscale, and UV irradiation mainly caused changes in the molecular binding mode. The results provide important knowledge for analyzing archeological lacquer samples and for developing lacquer-based materials. This work also demonstrates the utility of the complementary use of XANES, EXAFS, FT-IR, SAXS, and SANS for nondestructive analysis of black lacquer in precious cultural relics.