The biosynthetic pathway of oleuropein (from 7-ketologanin, oleoside-11-methyl ester, 7-β-1-d-glucopyranosyl-11-methyl oleoside, and ligstroside to oleuropein) was investigated in two fruit species of Oleaceae, namely, Arbequina and Hojiblanca. Main oleuropein precursors and their metabolites, produced by the enzymatic hydrolysis mediated by β-glucosidase, were identified and quantified to establish the oleuropein transformation pathway. Changes in the concentration of these compounds were measured by direct control of in vivo fruit tissue during their ripening. High contents of aglycones at the initial stage of the process were caused by the high activity of β-glucosidase, which supports that oleuropein biosynthesis is coupled with enzymatic hydrolysis, producing its aglycone form. The low oleuropein content at this initial stage was caused by the imbalance between catabolic and anabolic pathways, favoring the former ones. Once the main polyphenol synthesis phase was completed, the biosynthetic capacity diminished and the content of all compounds decreased. Mass balance revealed that precursors of oleuropein, which are rapidly transformed by β-glucosidase and esterases, scarcely contributed to the accumulation of oleuropein. The biosynthetic pathway proposed by Damtoft applies for both varieties, but our study reveals that the β-glucosidase enzyme is involved in oleuropein synthesis. This enzyme shows high substrate specificity to oleuropein, which consequently is degraded to its aglycone form, with diminished efficacy of oleuropein biosynthesis. Different enzymatic activities of varieties will result in oleuropein accumulation and metabolic transformation of phenols.