Detection of polystyrene beads with a diameter of 100 nm was conducted using an optical disk drive tester equipped with a laser of 405 nm wavelength and an objective lens with a numerical aperture of 0.65. The polystyrene beads were used to mimic the influenza virus. A grooved disk with a (ZnS)₈₅(SiO₂)₁₅/SiO₂ layered surface structure was used for the detection. The detection of influenza viruses (A/Udorn/307/1972) with Au nanoparticles was also demonstrated using the optical disk drive tester. In this case, a grooved disk with an indium tin oxide (ITO) film was used. The ITO film functioned both to tune the reflectance of the disk and as an electrically conductive layer for scanning electron microscopy. In both cases, the target substances were successfully recognized in a single scan with a high scanning speed of 4.9 m/s. The results indicate that this optical disk system can be used to detect 100 nm scale substances like influenza viruses, which are smaller than the diffraction limit of the system.