Abstract Background Diet has a direct impact on our health and as such is an important leverage in public health prevention strategies. Nutri-Score is a scientifically validated 5-color front-of-pack nutrition label based on a nutrient profile called FSAm-NPS that was proposed as a tool to help people choose healthier foods. It has been adopted by several European countries, but remains optional under current EU labeling regulation (under revision). This study aimed to provide insights into the relevance of the FSAm-NPS to characterize healthier foods by studying associations with a range of biomarkers reflecting pathways underlying nutrition-health associations. Methods This analysis included healthy participants from the EPIC cohort study (inclusion in 1992-2000, 8 European countries) for which blood biomarkers were measured from baseline samples in several case-control studies. Food intakes were assessed using country-specific dietary questionnaires. The FSAm-NPS was calculated for each food based on its 100g content in energy, sugar, saturated fatty acid, sodium, fibre, protein, and fruit and vegetables. Multi-adjusted linear models were computed. Results Consuming foods with higher FSAm-NPS scores (lower nutritional quality) was positively associated with the concentration of C-peptide (n = 788, β = 0.02, P = 0.04) and C-reactive protein (n = 899, β = 0.04, P = 0.04) and with a multi-marker profile characterized by apolipoprotein B, total and LDL cholesterol concentrations derived from principal component analysis (n = 498, lsmeans = -0.35 in quintile 1 and -0.03 in quintile 5, P = 0.02). Conclusions In this European context, higher concentrations of blood biomarkers reflecting hyperinsulinism, inflammation and dyslipidemia were observed in participants consuming foods and beverages with higher FSAm-NPS scores (lower nutritional quality/less favourable Nutri-Score). This adds to the evidence on the relevance of Nutri-Score as a public health tool to help people eat healthier food products. Key messages • The consumption of foods with a lower nutritional quality as graded by the Nutri-Score was associated with concentrations of blood biomarkers reflecting hyperinsulinism, inflammation and dyslipidemia. • This adds to the evidence supporting the relevance of the Nutri-Score as a complementary tool to dietary guidelines to help people choose healthier foods and beverages.