<p>Previous studies investigating the influence of hot spring bathing on sleep quality have predominantly focused on the short-term effects through questionnaire surveys without blood collection for biochemical tests. Here, we undertook a comprehensive investigation of the long-term health effects of hot spring bathing among the residents of Hot Spring Village. A total of 140 participants were enrolled, and their demographic characteristics and the patterns of hot spring bathing were obtained via face-to-face interview, and sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). Moreover, the blood samples of the participants were collected for biomarker detection with the ELISA assay. Logistic regression analysis was applied to evaluate the relationship between sleep quality and serum biomarkers among long-term hot spring bathers. In individuals aged 65 and older, the results demonstrated that hot spring bathing (<i>OR</i> = 0.18, 95%<i>CI</i>: 0.05–0.68), particularly with a length of ≥ 30&#xa0;min (<i>OR</i> = 0.10, 95%<i>CI</i>: 0.02–0.53) and a frequency of ≥ 3 times/week (<i>OR</i> = 0.07, 95%<i>CI</i>: 0.01–0.32) were significant protective factors for good sleep quality (<i>P</i> &lt; 0.05). Furthermore, reduced TNF-α (<i>OR</i> = 1.03, 95% <i>CI</i>: 1.01–1.06) and increased 5-HT levels (<i>OR</i> = 0.98, 95% <i>CI</i>: 0.97–0.99) were associated with good sleep quality. Interestingly, for the first time, we observed that the TNF-α significantly decreased (<i>P</i> &lt; 0.05) in the bathing group, along with an increasing trend of 5-HT and BDNF. Moreover, among participants in this group who reported good sleep quality, there was a notably significant decrease in TNF-α and an increase in 5-HT levels as well. These findings suggested that long-term hot spring bathing is associated with good sleep quality through the alteration of TNF-α and 5-HT levels, which could be potential biomarkers for future investigation on the health-promoting effects of bathing.</p>