Background: In clinical practice, the cases with bacterial infection caused by titanium implants and bacterial biofilm formation on the surface of titanium materials implanted into human body can often be observed. Thus, this study aimed to demonstrate whether the mixed biofilm ofStaphylococcus aureus/Escherichia colican be formed on the surface of titanium material through in vitro experiments and its formation rules. Methods: The titanium plates were put into the well containingS aureusor/andE coli. Bacterial adhesion and biofilm formation were analyzed by crystal violet, XTT method, confocal laser scanning microscopy, and scanning electron microscopy. Results: The results of bacterial adhesion in each group at 6-72 hours showed that the number of bacterial adhesion in each group was increased with the extension of time and reached to the highest level at 72 hours. Moreover, the biofilm structure in theS aureus-E coligroup was significantly more complex than that of the simpleS aureusgroup orE coligroup, and the number of bacteria was also significantly increased in theS aureus-E coligroup. Conclusion: Those data provide a laboratory basis for the prevention and treatment of mixed infection of subsequent biological materials.