The Nankai Trough hosts diverse fault slip modes, ranging from slow slip events to megathrust earthquake ruptures. We performed laboratory friction experiments on samples collected by the Integrated Ocean Drilling Program offshore Kii Peninsula, Japan. This study systematically investigates the effect of effective normal stress on frictional strength and the velocity-dependence of friction for natural fault zone and wall rock samples collected from depths of 270 to ∼450 meters below seafloor (mbsf), and over a range of shearing velocity spanning from 0.01–30 μm/s. In addition, cohesive strength was determined before and after each velocity step experiment while the sample was unloaded. We tested both powdered and intact specimens at estimated in-situ effective stresses, as well as at higher stresses representing deeper portions of the megasplay fault (Sites C0004, C0010) and the frontal thrust zone (Sites C0007, C0006). The apparent coefficient of sliding friction μs varies between 0.22–0.53 and correlates inversely with clay content. Direct measurements of cohesive strength show that on average 11% of the residual shear strength, and up to ∼30% for some specimens, can be attributed to cohesion. Friction coefficient slightly decreases as a function of increasing effective normal stress, attributed to a decreasing proportion of cohesive strength. The lowest μs values are observed for samples from the frontal thrust zone Site C0007 and the megasplay fault Site C0010. All samples show a combination of velocity-strengthening and velocity-weakening behavior, but intact samples tested under in-situ effective normal stress and low shearing velocities (<∼1 μm/s) exhibit consistently large velocity-weakening frictional behavior. The observed velocity-weakening behavior is related to the induration state of the material, which affects the real area of contact along shear surfaces. This is supported by direct measurements of cohesive strength, showing that higher cohesion values in intact samples correspond with a more pronounced evolutionary effect in velocity step tests that, in turn, map to lower values of the rate-dependent friction parameter a−b. We propose that fault zones in the Nankai subduction zone are likely to be velocity-weakening, and thus potentially able to host the nucleation of unstable slip, from seismogenic depths to the seafloor. We also find that testing disaggregated fault zone samples and employing effective stresses exceeding those in-situ lead to overestimation of a−b, emphasizing the importance of testing intact samples under in-situ conditions in laboratory friction studies.