This study investigated the effects of water deficit stress on the growth, physiological, and biochemical parameters of four Petunia cultivars: 1. Iranian Petunia (P1), 2. Hybrid Supercascade White Petunia (P2), 3. Hybrid Grandiflora Frost Blue Petunia (P3), and 4. Hybrid Grandiflora Crimson Star Petunia (P4) under different levels of water deficit (90%, 60%, and 30% of field capacity). The experiment was conducted in a factorial design with four replications in a completely randomized layout. The results showed that water deficit stress significantly reduced the fresh and dry weight of shoots and roots. The greatest reductions in shoot and root fresh and dry weights were observed in cultivars P4 and P3. Under severe stress conditions, stem height in cultivars P1 and P2 decreased by 40.4% and 43.3%, respectively. Moreover, the number of lateral branches in cultivars P1 and P3 increased under moderate water deficit stress (by 24.4% and 42.9%, respectively), but significantly declined under severe stress. Water deficit stress also significantly reduced root diameter in cultivar P3 (by 48%). Severe water stress decreased both flower diameter and the number of flowers in all four cultivars, with the greatest reduction in flower diameter (22.97%) observed in cultivar P4 and the highest reduction in flower number (72.3%) recorded in cultivar P1. Electrolyte leakage increased under severe water deficit stress, with cultivar P4 exhibiting the highest electrolyte leakage (36.1%). Leaf relative water content decreased under drought conditions, with the greatest reduction (24.4%) observed in cultivar P4. Proline content reached its highest level in cultivar P1 under severe stress conditions (2.24 µmol g⁻¹ fresh weight). Total carbohydrate concentration increased in cultivars P1 and P2 under severe stress (by 21.6% and 19.5%, respectively). Chlorophyll a and b contents decreased under drought conditions, with cultivar P4 showing the lowest chlorophyll b content (0.53 mg g⁻¹ fresh leaf weight) under severe stress. Superoxide dismutase (SOD) activity increased under moderate stress in cultivars P1, P2, and P3 (by 34.45%, 52.5%, and 24.9%, respectively), but showed no significant changes under severe stress. Catalase (CAT) activity increased in cultivar P1 (by 29.5%), while it decreased in the other three cultivars. Peroxidase (POD) activity was highest in cultivar P1 under non-stress conditions (5.65 units g⁻¹ fresh weight) and further increased (by 9.6%) under water deficit stress. Pearson correlation analysis revealed that proline and total carbohydrate contents were positively correlated with antioxidant enzyme activities, and chlorophyll a and b were closely associated with each other. Overall, cultivar P1 exhibited the highest drought tolerance, followed by cultivars P2 and P4, respectively.