Objective: Footbaths are employed in nursing practice in East Asian countries. Assessing the effects of footbaths based on the findings of multiple physiological responses is relevant to ensuring evidence-based nursing practice. This study aimed to investigate the effects of thermal stimulation by a footbath on prefrontal cortex activity and autonomic nervous function. Methods: This study was a randomized controlled crossover trial conducted on healthy participants from a college student population. Each participant underwent a footbath in warm water and a control condition for 10 min on two different days. The order of the two treatments was randomized. The oxygenated hemoglobin concentration in the prefrontal cortex, laterality scores of the oxygenated hemoglobin concentration changes, and autonomic responses were evaluated. Results: A total of 17 healthy participants were recruited. A footbath in warm water significantly reduced both the prefrontal cortex and sympathetic nerve activities 7 min after the start of the footbath compared with the control condition. Moreover, a footbath in warm water tended to activate the left prefrontal cortex rather than the right prefrontal cortex. Conclusions: The changes in the prefrontal cortex activity and autonomic nervous function were associated with the relaxing effect of the thermal stimulation by the footbath. Furthermore, this effect was highest at 7 min after the start of the footbath. This study has the potential to contribute to the evidence-based use of footbaths.