In this study, we investigate the microscale structure of shark skin denticles at abdomen (A) and fin (F) locations, analyze the roughness and wetting properties related to their microstructures, and evaluate the effect of the surface properties on early bacterial attachment and biofilm formation. Microstructural analysis by scanning electron microscopy and confocal laser scanning microscopy confirmed the length (A: 165–180 μm vs. F: 145–165 μm), width (A: 86–100 μm vs. F: 64–70 μm), height (A: 10.5–13.5 μm vs. F: 6.2–8.8 μm), and density (A: 110–130 denticles/mm2 vs. F: 80–130 denticles/mm2) of the denticles. The results showed that the roughness and hydrophobicity properties were affected with slight differences in the microscale architecture. The denticles with a larger width, higher ridge, and denser overlap provided a rougher and more hydrophobic surface. The microscale structure not only affected surface properties but also the biological attachment process. The microscale topography of shark skin slightly promoted bacterial attachment at an early stage, but prevented bacteria from developing biofilms. This systematic investigation provides insights into the effects of the surface topography of shark skin on its anti-fouling mechanism, which will enable the future development of various products related to human activity, such as healthcare products, underwater devices and applications, and water treatment applications.