Staphylococcus aureus has posed a huge threat to human health and the economy. Oleuropein has antibacterial activities against various microorganisms but research on its effect on the S. aureus biofilm is limited. This research aimed to estimate the antibiofilm activities of oleuropein against S. aureus. The results suggest that the minimum inhibitory concentration of oleuropein against S. aureus ATCC 25923 was 3 mg/mL. The biomass of biofilms formed on the microplates and coverslips and the viability of bacteria were significantly reduced after the treatment with oleuropein. The scanning electron microscopy observation results indicated that the stacking thickness and density of the biofilm decreased when S. aureus was exposed to oleuropein. It had a bactericidal effect on biofilm bacteria and removed polysaccharides and proteins from mature biofilms. The effects of oleuropein on the biofilm could be explained by a reduction in bacterial secretion of extracellular polymeric substances and a change in bacterial surface hydrophobicity. Based on the above findings, oleuropein has the potential to be used against food pollution caused by S. aureus biofilms.