Metabolome-based biomarkers contribute to identify mechanisms of disease and to a better understanding of overall mortality. In a long-term follow-up subsample (n = 1878) of the PREDIMED trial, among 337 candidate baseline plasma metabolites repeatedly assessed at baseline and after 1 year, 38 plasma metabolites were identified as predictors of all-cause mortality. Gamma-amino-butyric acid (GABA), homoarginine, serine, creatine, 1-methylnicotinamide and a set of sphingomyelins, plasmalogens, phosphatidylethanolamines and cholesterol esters were inversely associated with all-cause mortality, whereas plasma dimethylguanidino valeric acid (DMGV), choline, short and long-chain acylcarnitines, 4-acetamidobutanoate, pseudouridine, 7-methylguanine, N6-acetyllysine, phenylacetylglutamine and creatinine were associated with higher mortality. The multi-metabolite signature created as a linear combination of these selected metabolites, also showed a strong association with all-cause mortality using plasma samples collected at 1-year follow-up in PREDIMED. This association was subsequently confirmed in 4 independent American cohorts, validating the signature as a consistent predictor of all-cause mortality across diverse populations.