<p>Biofilms of <i>Staphylococcus aureus</i> pose significant challenges in various environments due to their resilience against antimicrobial agents. This study evaluated the antimicrobial efficacy of single disinfectant including 0.25% calcium hydroxide, 0.25% sodium hypochlorite, 0.25% deoxycholate, 0.25% poly(vinylpyrrolidone)-iodine complex and combination (0.25%) disinfectant suspensions against <i>S. aureus</i> (SA^ATCC and SA^CCARM) biofilms on metal disc surfaces. The results revealed that all treatments significantly reduced biofilm formation compared to the untreated control, with the combination treatment exhibiting the most potent and sustained anti-biofilm activity. After 4&#xa0;h of incubation, biofilm cells were undetectable under the combination treatment (&lt; 20&#xa0;CFU/mL), indicating complete eradication. Crystal violet staining and biofilm formation index analysis confirmed that the combination treatment resulted in the lowest biofilm biomass. Additionally, extracellular DNA and polysaccharide levels were markedly reduced, further supporting the disruption of biofilm integrity. Gene expression analysis demonstrated a significant downregulation of biofilm-associated genes, including <i>norB</i>, <i>clpP</i>, <i>clpX</i>, and <i>dnaK</i>, under all treatments, with the combination treatment showing the highest inhibitory effect. Transmission electron microscopy analysis revealed severe morphological alterations and biofilm dispersal in the combination-treated samples. Collectively, these findings suggest that the combination disinfectant formulation is the most effective strategy for eradicating <i>S. aureus</i> biofilms, offering a promising approach for biofilm control in medical, industrial, and environmental applications.</p>