<p>Multi-species biofilms present a greater threat to the dairy industry than single-species biofilms due to their complex characteristics. This study examined the formation of biofilms by dairy contaminants <i>Staphylococcus aureus</i> and <i>Pseudomonas fluorescens</i> (single/co-culture) on stainless steel, and their resistance to chlorine dioxide and quaternary ammonium compounds. The findings indicated that the dual-species biofilm exhibited significantly higher biomass, cell activity, and extracellular polymeric substances production than single-species biofilms, along with a denser biofilm structure in dual-species biofilm and increased resistance to both disinfectants. Remarkably, relative quantification analysis using RT-qPCR revealed an interaction: the presence of <i>P. fluorescens</i> markedly upregulated the <i>ica</i>A and <i>ica</i>D genes in <i>S. aureus</i>. This genetic modulation was directly correlated with increased production of polysaccharide intercellular adhesion and EPS, which are crucial for biofilm maturation and structural integrity. This study offers a theoretical foundation for the hazard control of multi-species biofilms in the dairy industry.</p>