This study investigates the combined effect of salicylic acid (SA) and sodium nitroprusside (SNP), a nitric oxide (NO) donor, on the antioxidant defense system of two upland cotton genotypes (Gossypium hirsutum L. Agdash-3 and AP-317) under hydroponic conditions. Seedlings were treated with 0.1 mM SA and two concentrations of SNP (0.1 and 2.5 mM), and biochemical responses were assessed after two weeks of growth. The activities of catalase, peroxidase, and polyphenol oxidase, as well as nitric oxide and proline contents were measured in the leaves. The results revealed genotype-specific responses to the treatments. In Agdash-3, antioxidant enzyme activities and proline levels increased proportionally with SNP concentration, indicating synergistic interactions between SA and SNP. In contrast, AP-317 showed higher sensitivity to SA but reduced proline accumulation in response to low SNP concentrations. The findings demonstrate that the interplay between SA and SNP modulates the antioxidant response in a genotype-dependent manner, suggesting potential applications in enhancing stress tolerance in cotton through phytohormonal and NO-based treatments.