Exposure to natural sounds is known to induce feelings of relaxation; however, only few studies have provided scientific evidence on its physiological effects. This study examined prefrontal cortex and autonomic nervous activities in response to forest sound. A total of 29 female university students (mean age 22.3 ± 2.1 years) were exposed to high-resolution sounds of a forest or city for 60 s, using headphones. Oxyhemoglobin (oxy-Hb) concentrations in the prefrontal cortex were determined by near-infrared spectroscopy. Heart rate, the high-frequency component of heart rate variability (which reflects parasympathetic nervous activity), and the ratio of low-frequency to high-frequency (LF/HF) components (which reflects sympathetic nervous activity) were measured. Subjective evaluation was performed using the modified semantic differential method and profiles of mood states. Exposure to the forest sound resulted in the following significant differences compared with exposure to city sound: decreased oxy-Hb concentrations in the right prefrontal cortex; decreased ln(LF/HF); decreased heart rate; improved feelings described as “comfortable,’’ “relaxed,” and “natural”; and improved mood states. The findings of this study demonstrated that forest-derived auditory stimulation induced physiological and psychological relaxation effects.