This study aimed to clarify the effects of differences in the wood subflooring and finishing on the physiology and psychology of walking barefoot. We measured surface electromyography (EMG) at six lower limb locations during walking on four different model floors, combining two types of flooring base materials with different stiffness and two types of floor finishes with different surface properties. We then conducted a psychological evaluation of the walking feelings. The 24-mm thick high-stiffness plywood subflooring had a significantly lower integrated EMG for the biceps femoris muscle while walking than the low-stiffness joist and 15-mm thick plywood subflooring, indicating less stress on the feet. Surface consolidated Japanese cedar, sugi (Cryptomeria japonica) wood finishing with the rough surface tended to have a lower surface integrated EMG during walking than ceramic-coated Japanese cypress, hinoki (Chamaecyparis obtusa) wood finishing with a smooth surface. Smooth finishing materials were rated as easier to walk on in the impression evaluation, which was contrasted with the surface EMG results. EMG is considered to be better suited for predicting the accumulation of fatigue due to prolonged walking, which is difficult to recognize after a short period of walking.