Background: Dietary biomarkers may complement dietary intake assessment made by dietary questionnaires. We developed an a-posteriori dietary biomarkers score based on Mediterranean diet food groups and evaluated its association with mortality. Methods: 642 participants (56% female), aged >= 65 years, with complete data on dietary biomarkers were followed during 20 years in the InCHIANTI cohort study (Tuscany, Italy). The main outcomes were all-cause, cardiovascular, and cancer mortality. Dietary biomarkers were selected from literature and from correlation analyses with dietary intakes of Mediterranean diet food groups in the study. The baseline levels of the following dietary biomarkers were chosen: urinary total polyphenols and resveratrol metabolites, and plasma carotenoids, selenium, vitamin B12, linolenic, eicosapentaenoic and docosahexaenoic acids, and the mono-unsaturated/saturated fatty acid ratio. Associations of the Mediterranean diet score using dietary biomarkers and a validated food frequency questionnaire (FFQ) (as tertiles) with mortality were assessed through Cox regression. Results: During the 20-year follow-up [median (Q1-Q3), 14 (8-18) years], and 435 deaths occurred (139 from cardiovascular diseases and 89 from cancer-related causes). In the fully adjusted models, the dietary biomarker-Mediterranean diet score was inversely associated with all-cause (HRT3vs.T1 0.72; 95%CI 056-0.91) and cardiovascular (HRT3vs.T1 0.60; 95%CI 0.38-0.93), but not with cancer mortality. Associations between the FFQ-Mediterranean diet score and mortality were not statistically significant. Conclusions: A greater adherence at baseline to a Mediterranean diet assessed by a dietary biomarker score was associated with a lower risk of mortality in older adults during a 20-year follow-up. The measurement of dietary biomarkers may contribute to guide individualized dietary counseling to older people.