Introduction: The stringent response is a bacterial adaptation mechanism triggered by stress conditions, including nutrient limitation. This response helps bacteria survive under harsh conditions, such as those encountered during infection. A key feature of the stringent response is the synthesis of the alarmone (p)ppGpp, which influences various bacterial phenotypes. In several bacterial species, stringent response activation significantly affects biofilm formation and maintenance. Methods: Clinical specimens were collected from multiple hospitals in Baghdad, Iraq. Staphylococcus aureus was identified using conventional biochemical tests. The PCR technique was applied to detect mecA, icaA, and icaD genes, while the Vitek 2 compact system confirmed Methicillin sensitivity in mecA-negative isolates. Biofilm intensity of all S. aureus isolates was assessed under normal and starved conditions. Additionally, the gene expression levels of icaA and icaD were measured in five MRSA and five MSSA strains under both conditions. Results: The mecA, icaA, and icaD genes were detected in 94%, 96.3%, and 100% of S. aureus isolates, respectively. Biofilm production analysis showed that 24% of isolates were strong producers, 49% were moderate producers, and 9% were weak producers. Statistical analysis indicated that biofilm intensity significantly decreased under nutrient limitation (p < 0.0001) compared to normal conditions across all isolates. Furthermore, icaA and icaD genes were upregulated under stringent response conditions, regardless of Methicillin resistance status. Conclusion: The stringent response influences S. aureus biofilm formation, with biofilm intensity decreasing under nutrient-limited conditions. However, the upregulation of icaA and icaD genes suggests a regulatory role of the stringent response in biofilm-related gene expression. These findings highlight the potential impact of stress adaptation mechanisms on bacterial persistence and pathogenicity.