Strong ground motions of the 2016 southeast off‐Mie, Japan, earthquake ( 6.5) were recorded by the eastern Dense Oceanfloor Network system for Earthquakes and Tsunamis (DONET1) seafloor observation network in the Nankai subduction zone. The observed values in peak ground velocity and acceleration response spectra (SA) at periods of 0.5–8 s at seafloor stations were much larger than those predicted by the empirical attenuation relationship. The observed values were primarily caused by the strong site amplification effect due to the subsurface structure, such as an accretionary wedge and shallow soft sediments. On the other hand, the observed values in peak ground acceleration and SA at periods of 0.12 and 0.25 s follow the empirical relationship, which is inconsistent with the strong site amplification effect suggested by spectral inversion. To understand this inconsistency, we investigate the nonlinear soil response at DONET1 stations during this event using time–frequency analyses and S‐wave horizontal‐to‐vertical spectral ratios. The analyses indicate that a significant nonlinear soil response occurred at several stations. This nonlinear soil behavior partially contributed to the suppression of the amplification of short‐period ground motions at DONET1 stations during the 2016 southeast off‐Mie earthquake.