Mature `Picual' olive ( Olea europaea L.) trees growing in two different localities of Córdoba and Jaén provinces, southern Spain, were subjected to annual applications of 0, 0.12, 0.25, 0.50, or 1.0 kg N/tree in the Cordoba's experiment, and to 0 or 1.5 kg N/tree in the Jaén's experiment. Nitrogen was applied 50% to the soil and 50% through foliar application in Córdoba, and 100% to the soil in Jaén. Three years after the initiation of treatments, when the trees showed differences among them in nitrogen content, fruit were sampled at maturity from each experimental tree during six consecutive seasons to determine the effect of nitrogen fertilization on olive oil quality. Tree nitrogen status was always above the threshold limit for deficiency even in control trees, indicating that most treatments caused nitrogen over fertilization. Nitrogen in excess was accumulated in fruit and, consequently, polyphenol content, the main natural antioxidants, significantly decreased in olive oil as nitrogen increased in fruit. The decrease in polyphenols induced a significant decrease in the oxidative stability of the oil and its bitterness. Tocopherol content, on the contrary, increased with nitrogen application, mainly by an increase in α-tocopherol, the main component in the olive oil. No effect was found on pigment content, particularly carotenoid and chlorophyllic pigments, neither on fatty acid composition.