Aims Over-the-counter mouthwash comprises part of routine oral care for many; however, potential adverse effects of the long-term daily use have not been evaluated. Most mouthwash contain antibacterial ingredients, which could impact oral microbes critical for nitric oxide formation, and in turn predispose to metabolic disorders including diabetes. Our aim was to evaluate longitudinally the association between baseline over-the-counter mouthwash use and development of pre-diabetes/diabetes over a 3-year follow-up. Materials and methods The San Juan Overweight Adults Longitudinal Study (SOALS) recruited 1206 overweight/obese individuals, aged 40–65, and free of diabetes and major cardiovascular diseases; 945 with complete follow-up data were included in the analyses. We used Poisson regression models adjusting for baseline age, sex, smoking, physical activity, waist circumference, alcohol consumption, pre-hypertension/hypertension status; time between visits was included in the models as an offset. Results Many participants (43%) used mouthwash at least once daily and 22% at least twice daily. Participants using mouthwash ≥ twice daily at baseline, had a significantly elevated risk of pre-diabetes/diabetes compared to less frequent users (multivariate IRR = 1.55, 95% CI: 1.21–1.99), or non-users of mouthwash (multivariate IRR = 1.49; 95% CI: 1.13–1.95). The effect estimates were similar after adding income, education, oral hygiene, oral conditions, sleep breathing disorders, diet (processed meat, fruit, and vegetable intake), medications, HOMA-IR, fasting glucose, 2hr post load glucose or CRP to the multivariate models. Both associations were also significant among never-smokers and obese individuals. Mouthwash use lower than twice daily showed no association, suggesting a threshold effect at twice or more daily. Conclusions Frequent regular use of over-the-counter mouthwash was associated with increased risk of developing pre-diabetes/diabetes in this population.