Staphylococcus aureus (S. aureus) is a common bacterium that can cause a wide range of infections in humans. The growth of these bacteria is inhibited by strategies using antibiotics and/or nanomaterials. However, nanomaterials and antibiotics can cause cytotoxicity and antibiotic resistance. Recent research has focused on achieving mechanical inhibition of bacterial growth through the surface nanostructure. Antibacterial use of nanostructured surfaces does not need antibiotics. Meanwhile, transparent films are necessary in the medical, optical, and automotive industries. In this study, we proposed a flexible, transparent, antibacterial, and antifogging film with a nanostructured surface. The bactericidal and bacteriostatic effects were observed on the arrayed nanopillars according to spacing. For S. aureus, bactericidal effect was observed at a nanopillar spacing of 300 nm, and bacteriostatic effect was observed at 500−1000 nm. Furthermore, the antibacterial rate was improved by 94.9% through MPC polymer coating at the surface. In addition, the MPC coating maintained an antibacterial rate of 86.6 and 64.1% for 7 and 14 days, respectively. The hydrophilic MPC-coated surface has properties of antifogging and transparency for potential usage in optical devices. This study provides insight into the potential of nanostructured surfaces using a dual strategy to prevent bacterial infections.