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Abstract Wood is a raw material that is used not only in the production of structural members for various buildings, but also in the interior finishes that are directly seen and touched by the occupants. Wood has also been recognized as a human-friendly material, but few studies have experimentally confirmed the physiological benefits it brings to humans. Therefore, the aim of the present study was to investigate the physiological effects of visual stimulation with wood. Two types of full-scale square, wooden-wall images composed of vertically or horizontally arranged lumber, were prepared using computer graphics and projected onto a large display to create the visual stimuli, and a gray image was also prepared as a control. Twenty-eight female Japanese university students participated in the study. The participants initially spent 60 s viewing the gray background (rest period) and then observed each of the wooden-wall images and the gray image separately in a random order for 90 s each. During the visual stimulation, the oxyhemoglobin (oxy-Hb) concentration as an indicator of prefrontal brain activity and heart rate variability as an indicator of autonomic nervous activity were continuously measured in each participant. Subjective evaluation of each visual stimulus was then performed using a modified semantic differential method and the Profile of Mood States 2nd Edition test. It was found that visual stimulation with either of the wooden interior wall images induced a significant decrease in oxy-Hb concentration in the left and right prefrontal cortex compared with the gray image. Furthermore, the subjective evaluation showed that the wooden-wall images provided a significantly more “comfortable,” “relaxed,” and “natural” impression than the gray image and decreased the negative mood states, with the vertically arranged wooden-wall image having a more positive effect than the horizontally arranged image.