Introduction: Medical students are particularly susceptible to nutritional deficiencies and mental health problems due to intensive study demands, stress, and lifestyle factors. Vitamin D3, vitamin B12, and folate deficiencies have been implicated in mental well-being, although evidence remains inconsistent. Objective: To assess the prevalence of vitamin D3, vitamin B12, and folate deficiencies among medical students at the University of Warmia and Mazury in Olsztyn, and to explore associations between serum concentrations of these vitamins, lifestyle factors, and self-reported well-being. Materials and Methods: The study included 97 medical students. Serum vitamin concentrations were measured using electrochemiluminescence immunoassay. Well-being was assessed with the WHO-5 Well-Being Index. Group comparisons were performed using non-parametric tests, and a Poisson regression model was applied as an exploratory analysis to examine associations between selected lifestyle factors and well-being. Results: Vitamin D3 deficiency was observed in 78% of students, folate deficiency in 20%, and vitamin B12 deficiency in 8%. In unadjusted analyses, differences in serum vitamin D3 and vitamin B12 concentrations were observed between students with lower and higher self-reported well-being, whereas folate concentrations did not differ. However, after correction for multiple testing using the Benjamini–Hochberg procedure, none of these associations remained statistically significant. Exploratory regression analysis suggested that physical activity and gender may be associated with well-being, while no association with vitamin D3 supplementation was observed. Conclusions: Vitamin D3, vitamin B12, and folate deficiencies were common among medical students. Exploratory analyses suggested differences in vitamin D3 and vitamin B12 concentrations across well-being groups; however, these findings did not remain significant after correction for multiple testing and should be interpreted with caution. Overall, the results indicate that lifestyle-related factors, particularly physical activity, may play a more prominent role in student well-being than serum vitamin concentrations alone. Further longitudinal studies are required to clarify these relationships.