Olive anthracnose caused by the hemibiotrophic fungal pathogen Colletotrichum acutatum is a serious threat to the olive sector. Olive oil and fruit production is severely constrained by Colletotrichum spp. infection, being C. acutatum the most distributed pathogen in Portuguese olive orchards. To understand the impact of C. acutatum on phenylpropanoids biosynthesis, the enzyme activity, phenolic compounds, ortho-diphenols, and flavonoids content were determined and correlated with the expression of gene encoding key enzymes within phenylpropanoids metabolism in susceptible and tolerant olive fruits, during maturation and when infected with C. acutatum. Differences between cultivars was observed, the tolerant olive cv. Picual presented a higher basal value and a stable phenolic content throughout the infection process, supporting its high C. acutatum tolerance, whereas in the susceptible olive cv. Galega these secondary metabolites were significantly increased only after the elicitation with C. acutatum.