Blood oranges are increasingly cultivated worldwide as consumers become more aware of the health benefits of their nutraceutical properties and natural antioxidants, specifically polyphenols and anthocyanins. The amounts of these compounds in the fruit mostly depend on the cultivar, rootstock, maturity stage, and environmental conditions. This work focused on the study of the qualitative features of numerous blood orange cultivars grown in three different environments in Spain and Italy. The aim of the work was to investigate the accumulation of primary and secondary metabolites, including bioactive compounds, and to characterize fruit qualitative traits at the time of harvest. Simple sugars were identified and quantified by liquid chromatography and organic acids, polyphenols, and flavonoids by spectrophotometric analysis. The antioxidant potential of the juice was assessed by ABTS, DPPH, and FRAP assays. Cultivation area affected juice color, with Moro and T. Ippolito being the varieties with the highest pigmentation. The cultivation area also determined the pattern of primary and secondary metabolite accumulation in the Tarocco lines. Furthermore, the antioxidant potential was influenced by the diverse environments. Principal Component Analysis highlighted three clusters, two overlapping clusters for the varieties grown in the two Spanish plots and a third clearly separated cluster for the genotypes grown in Italy. This study provides novel knowledge on primary and secondary metabolite accumulation in blood oranges, elucidating the role of genotype and environmental conditions on fruit quality.