Drought-induced stress represents one of the most economically detrimental natural phenomena impacting grapevine (Vitis vinifera) development, yield, and fruit characteristics. Also, auxin is one of the most important plant growth regulators that can reduce damage caused by stress in plants. In this study, the impact of exogenously sprayed auxin (0, 50, and 200 mg L−1) on growth, biochemical, and anatomical parameters was investigated in two grapevine varieties (cvs. ‘Rashe’ and ‘Fakhri’) under water deficit. According to our findings, water deficit led to a notable decrease in growth, protein content, and anatomical parameters; but significantly enhanced electrolyte leakage. Grapevines exposed to water deficit exhibited substantial increases in total phenolic compounds and antioxidant activity. Applying 50 mg L−1 napthalene acetic acid (NAA) reduced the effects of water deficit in both grapevine cultivars by decreasing electrolyte leakage (15% in ‘Rashe’ and 20% in ‘Fakhri’), and accumulating protein content (22% ‘Rashe’ and 32% ‘Fakhri’), total phenolic compounds (33% ‘Rashe’ and 40% ‘Fakhri’), and antioxidant capacity (11% ‘Rashe’ and 39% ‘Fakhri’); anantomical parameters were also improved. However, application of 200 mg L−1 NAA had adverse effects on growth and biochemical traits of grapevines, with a more pronounced impact on root growth and anatomical parameters compared to other NAA concentrations. In conclusion, the application of 50 mg L−1 NAA enhanced grapevine growth, enabling them to better thrive under water deficit.