Recent research suggests that museum visits can benefit psychological well-being by reducing symptoms of stress and anxiety. However, these reported relaxing effects remain inconsistent between studies. Shedding light on the underlying cerebral mechanisms of museum visits might support a better understanding of how it affects psychological well-being. This study aimed to investigate the prefrontal engagement evoked by artwork analysis during a museum visit and to determine if these prefrontal substrates are associated with the museum’s effect on psychological well-being in older adults. Nineteen adults aged between 65 and 79, toured a Baroque-style exhibit at the Montreal Museum of Fine Arts for approximately 20 minutes while equipped with a near-infrared spectroscopy system measuring the prefrontal cortex’s hemodynamic activity. For each painting, participants received the instruction to either (1): analyze the painting and produce a personal interpretation of its signification (analytic condition) or (2) visualize the painting without any specific thoughts (visualization condition). Questionnaires measuring stress, anxiety, and well-being were administered before and after the visit. Sixteen older women (71.5 ± 4 years) were included in the analyses. Results showed that, at the group level, the analytic condition was associated with an increased activation pattern in the left ventrolateral prefrontal region, typically related to attentional processes (not observed in the visualization condition). The activation associated with the analytic condition predicted pre-/post-visit reductions in self-reported anxiety and stress in the sample of older women. These observations suggest that the level of engagement of attentional processes during artwork analysis may play a major role in the effect of a museum’s visit on self-reported symptoms of anxiety.