In order to evaluate the physiological and biochemical responses of different ecotypes of caper (Capparis spinosa L.) seedlings to drought stress, an experiment was conducted under laboratory conditions in 2023 at the Seed Quality Analysis Laboratory of the Seed and Plant Certification and Registration Research Institute, Karaj, Iran, using a completely randomized design with three replications. In this study, seeds of nine native caper ecotypes collected from Ardabil Province (Moghan), Alborz (Karaj), Ilam Province (Mehran), North Khorasan Province (Bojnord), Kermanshah Province (Dalahoo), and Khuzestan Province (Ramhormoz, Hendijan, Mahshahr, and Ahvaz) were evaluated. Drought stress was imposed using polyethylene glycol (PEG 6000) at an osmotic potential of −6 bar. The results indicated that drought stress significantly increased malondialdehyde content from approximately 6.5 to 11.3 nmol g⁻¹ FW and hydrogen peroxide from 3.1 to 6.0 µmol g⁻¹ FW, while total chlorophyll content decreased from 0.35 to 0.21 mg g⁻¹ FW. In contrast, proline content (12.7–45.8 µg g⁻¹ FW), total phenolic content (2.6–3.6 mg gallic acid g⁻¹ FW), and antioxidant capacity (38–47% DPPH inhibition) showed significant increases under drought conditions. Comparison among ecotypes revealed that under non-stress conditions, the Karaj and Moghan ecotypes exhibited greater physiological stability, whereas under drought stress, the Mahshahr and Hendijan ecotypes showed the lowest oxidative damage indices and the highest accumulation of protective compounds, and were therefore identified as drought-tolerant ecotypes. Overall, the results highlight the key role of antioxidant defense mechanisms and osmotic adjustment in the adaptation of caper plants to drought stress conditions.