Background <p>Breast cancer (BC) is the primary cause of cancer-related death among women globally, highlighting the importance of the identification of novel biomarkers for early detection and prognosis. This study investigates the impact of NOS3 (endothelial nitric oxide synthase) and SOD1 (superoxide dismutase 1) gene polymorphisms on BC development, focusing on their associations with oxidative stress and tumor progression.</p> Methods <p>A case-control study involving 30 BC patients and 30 healthy controls was conducted. Mutations in NOS3 and SOD1 genes were identified using sanger sequencing. ELISA was used to measure oxidative stress markers such peroxynitrite (ONOO⁻) and superoxide dismutase (SOD). To investigate functional associations, protein–protein interaction networks were examined using the GENEMANIA database.</p> Results <p>The study identified 32 polymorphisms in the NOS3 gene and 16 polymorphisms in the SOD1 gene, as well as nine amino acids alterations predicted in SOD1 gene. In comparison to controls, BC patients had higher levels of ONOO- (0.095 ± 0.048 ng/L vs. 0.048 ± 0.057 ng/L, <i>p</i> &lt; 0.0001) and SOD (0.074 ± 0.033 ng/ml vs. 0.043 ± 0.045 ng/ml, <i>p</i> &lt; 0.001). GENEMANIA analysis showed the interactions among NOS3, SOD1, and oxidative stress-related genes, highlighting their significance in cellular redox balance.</p> Conclusions <p>These findings suggest that NOS3 and SOD1 polymorphisms may contribute to BC pathogenesis, as supported by the observed oxidative stress alterations (ONOO- and SOD levels). Additional validation in larger cohorts is needed to confirm their potential as biomarkers for risk evaluation and clinical advancement. The study enhances understanding of the genetic and oxidative stress mechanisms in cancer biology and identifies potential therapeutic targets for further investigation.</p>