Sunflower (Helianthus annuus L.), a globally significant oilseed crop, faces substantial yield losses due to drought stress, a major environmental constraint. In this study, the effects of nitric oxide (NO) to increase drought tolerance in four sunflower genotypes (resistant Irtysh, RAR 56 and sensitive Zarya, RAR 133) showing different stress responses were investigated. Conducted in a controlled hydroponic system, the experiment applied 100 µM NO under 12% polyethylene glycol (PEG)-induced drought, assessing growth, physiological, and biochemical parameters. PEG alone reduced shoot and root growth, relative water content (RWC), and ion levels (K, Ca, Mg, Na), while increasing oxidative stress markers (malondialdehyde (MDA), H2O2, •OH) and electrolyte leakage, particularly in sensitive genotypes. NO application, both alone and with PEG, significantly mitigated these effects, enhancing root fresh weight, RWC, and antioxidant enzyme activities (superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and glutathione reductase (GR)), while reducing reactive oxygen species (ROS) and lipid peroxidation. Resistant genotypes (Irtysh, RAR 56) exhibited superior stress amelioration. These findings highlight NO’s role as a signalling molecule in augmenting drought resilience through genotype-specific mechanisms. The differential responses among genotypes suggest opportunities for identifying genetic markers associated with NO-mediated drought tolerance, which could guide marker-assisted breeding programs. Additionally, integrating these insights with genomic editing techniques may accelerate the development of drought-resistant sunflower cultivars tailored for water-scarce regions. Future research should optimise NO delivery methods and evaluate field-scale efficacy to advance sustainable sunflower production in water-limited environments.