The characteristics of olive fruits, which strongly affect both the quality and quantity of olive oil, are affected by the position of the fruit in the canopy of olive trees. The present study aimed to understand the net effect of the position of the fruit in the canopy on the oil characteristics by comparing oils extracted from ripened olive fruits harvested at different heights in the tree canopy. Chemical analyses, measuring oil quality parameters, fatty acid composition and volatile compounds, showed higher values of acidic parameters, an increase in oxidative off-flavors, a decrease in olive oil aroma and a reduction in the concentration of oleic acid in oils extracted from fruits harvested from the higher canopy layers compared with those from the lower canopy layers. This suggested that lipid oxidation was greater in these oils compared with oils from fruit harvested from the lower layers. However, oils obtained from fruits from the higher layers exhibited higher antioxidant activities and contents of photoprotectors such as phenolics and carotenoids, which act as defense compounds under strong lighting conditions. These results suggest that environmental lighting conditions strongly affect oil quality and provide relevant information for methods of harvesting fruits destined for the production of high-quality olive oil.