Silver nanomaterials are often used as surface enhancement substrates even though silver is easily oxidized in air. In this paper, we investigate the influence of natural oxidation on the surface enhancement effect of silver nanoparticle (AgNP) films. The AgNP films were prepared by the vacuum thermal evaporation method, and the AgNP size range was 10–100 nm. Changes in the AgNP film thickness, surface morphology, particle size, and roughness with oxidation time were continuously measured with atomic force microscopy for 2 weeks. A detailed analysis of these parameters is presented. In addition, we used Rhodamine 6G to examine the surface enhancement effect of the AgNP films with different oxidation times. The results showed that the intensity of both the Raman and fluorescence signals increased and approximately periodically oscillated with increasing oxidation time. Here, we qualitatively discuss the mechanism of the influence of natural oxidation on the surface enhancement effect of silver nanostructures, which could provide a feasible way to improve the surface enhancement performance of silver nanomaterial substrates.