Olive is an essential industrial crop in the Mediterranean region with valuable economic and agricultural concerns. Despite its drought resistance, its productivity is restricted by extreme drought stress. Olive cultivars display considerable variation in response mechanisms to drought stress. Accordingly, the impact of mild and extreme water deprivation over two seasons compared to full irrigation requirements on growth and physiological characteristics of three diverse olive cultivars. Three olive cultivars, ‘Manzanillo’, ‘Eggizi-Shami’, and ‘Tofaahi’, were evaluated under three irrigation regimes 100% ETc, 75% ETc, and 50% ETc. Characteristics of shoot and root, as well as physio-chemical constituents, were determined. Besides, the gene expression of dehydration-responsive element binding (DREB), dehydrin (DHN), and catalase (CAT) genes in olive cultivars were explored under different irrigation regimes. The results indicated a substantial impact of irrigation level on all studied parameters. The mild and extreme drought stress treatments caused a gradual reduction in nitrogen, phosphorus, and potassium content, relative water content, root and shoot length, root and leaf numbers, branch count, and leaf area across both seasons. Conversely, proline content was considerably increased under drought treatments compared to well-watered conditions. Similarly, the assessed cultivars exhibited significant variation in all studied parameters, with ‘Eggizi-Shami’ demonstrating superiority. Under mild and extreme drought stress conditions, the cultivar ‘Eggizi-Shami’ displayed the highest proline content and most growth characteristics. Besides, the real-time quantitative PCR (RT-qPCR) analysis displayed significant alterations in gene expression of the tested three genes related to drought response (DHN, DREB, and CAT). The RT-qPCR analysis revealed that under drought stress conditions (75% and 50% ETc), ‘Eggizi-Shami’ exhibited higher expression compared to the other two cultivars (‘Tofaahi’ and ‘Manzanillo’). Combining the results of morphological and physiological parameters with gene expression analysis of drought-related genes can offer highly validated information about drought-tolerant olive cultivars. This integrated approach serves as an innovative methodology to identify and confirm genes involved in abiotic stress.