This study was aimed to clarify the physiological effects of visual stimulation using forest imagery on activity of the brain and autonomic nervous system. Seventeen female university students (mean age, 21.1 ± 1.0 years) participated in the study. As an indicator of brain activity, oxyhemoglobin (oxy-Hb) concentrations were measured in the left and right prefrontal cortex using near-infrared time-resolved spectroscopy. Heart rate variability (HRV) was used as an indicator of autonomic nervous activity. The high-frequency (HF) component of HRV, which reflected parasympathetic nervous activity, and the ratio of low-frequency (LF) and high-frequency components (LF/HF), which reflected sympathetic nervous activity, were measured. Forest and city (control) images were used as visual stimuli using a large plasma display window. After sitting at rest viewing a gray background for 60 s, participants viewed two images for 90 s. During rest and visual stimulation, HRV and oxy-Hb concentration in the prefrontal cortex were continuously measured. Immediately thereafter, subjective evaluation of feelings was performed using a modified semantic differential (SD) method. The results showed that visual stimulation with forest imagery induced (1) a significant decrease in oxy-Hb concentrations in the right prefrontal cortex and (2) a significant increase in perceptions of feeling “comfortable,” “relaxed,” and “natural.”