Olives are evergreen trees with a low growth rate that are cultivated in semi-tropical climates. The biochemical properties of three olive cultivars were explored under the foliar application of amino and organic acids in a two-factor factorial experiment based on a randomized complete block design with three replications in Rudbar County, Iran. The first factor was assigned to olive cultivar (‘Zard’, ‘Arbequina’, and ‘Manzanilla’) and the second factor to the foliar application of organic acids at 9 levels of control, arginine, glutamine, humic acid, fulvic acid, arginine + humic acid, arginine + fulvic acid, glutamine + humic acid, and glutamine + fulvic acid. The recorded traits included the Brix value, content of oil, protein, chlorophyll, carotenoid, anthocyanins, phenols, and the activity of antioxidant enzymes. The Results showed that cv. ‘Zard’ had the highest Brix value, fruit protein content, carotenoid, anthocyanins, and phenols, and cv. ‘Arbequina’ had the highest oil fraction. The Results of the simple effects of organic acids revealed that the trees treated with arginine + humic acid had the highest fruit protein content and total chlorophyll, and those treated with humic acid had the highest anthocyanin and phenol contents. Data on the interaction of ‘cultivar × organic acids’ showed that ‘Arbequina × glutamine’ had the highest oil content, ‘Manzanilla × fulvic acid’ and ‘Manzanilla × glutamine + fulvic acid’ had the highest fruit protein content, ‘Zard × humic acid’ had the highest phenol content, ‘Arbequina × arginine’ had the highest superoxide dismutase activity, and ‘Arbequina × glutamine + fulvic acid’ had the highest peroxidase activity. Finally, it can be concluded that Arbequina cultivar produced the most oil when foliar sprayed with glutamine.