This study investigated the interactions between Corynebacterium striatum and Staphylococcus aureus, two bacterial species commonly found in the human microbiota, particularly colonizing the skin and mucous membranes. Both organisms, however, are also capable of causing acute and chronic infections. While S. aureus is widely recognized as a clinically significant pathogen, C. striatum is frequently underestimated and often regarded as a contaminant—even when isolated in pure culture from nosocomial infections. The ability of these microorganisms to develop multidrug resistance and form biofilms complicates the management of the infections they cause. This study focused on the interaction between C. striatum and S. aureus, particularly the influence of the former on the pathogenic potential of the latter, emphasizing biofilm formation in S. aureus mutants deficient in the icaR and icaC genes. Antimicrobial susceptibility testing revealed that 85.7 % of the S. aureus strains were multidrug-resistant, with all strains resistant to β-lactam antibiotics. Additionally, 55.6 % of the strains produced strong slime on Congo Red agar, indicating a high potential for biofilm formation. In monomicrobial assays, both C. striatum and S. aureus exhibited enhanced adhesion to hydrophilic surfaces. In polymicrobial settings, C. striatum predominated in most cases: on glass surfaces, 70 % of biofilms were dominated by C. striatum, 20 % by S. aureus, and 10 % showed an even distribution. On polystyrene, 80 % of the biofilms were dominated by C. striatum, while 20 % were dominated by S. aureus. Analysis of extracellular polymeric substances (EPS) revealed distinct compositional profiles: C. striatum primarily produced proteinaceous matrices, whereas S. aureus biofilms were rich in polysaccharides. Ultrastructural examination showed that S. aureus formed dense clusters embedded in large amounts of EPS, while C. striatum biofilms exhibited lower EPS production overall. Furthermore, the effect of C. striatum-derived compounds on S. aureus biofilms was assessed. In 90 % of cocultured strains, a progressive decrease in sessile cell populations was observed, accompanied by an increase in planktonic cells. This finding suggests that C. striatum can disrupt the biofilm integrity of S. aureus, potentially modulating its pathogenic phenotype. In conclusion, the results demonstrate that C. striatum competes effectively with S. aureus for surface colonization and, under certain conditions, may induce a transition of S. aureus from a sessile to a planktonic state. These findings highlight the complexity of interspecies interactions in polymicrobial communities and suggest that C. striatum may play a modulatory role in S. aureus virulence. Such insights have important implications for the understanding and treatment of polymicrobial infections.