Moringa oleifera seed antimicrobial peptides MOp2 and MOp3 had good antibacterial effects on Staphylococcus aureus (S. aureus), but their effects on biofilm were still unclear. Therefore, crystal violet semi-quantitative method, fluorescence microscopy, enzymatic digestion experiment and molecular docking technology were used to study the effects of the newly identified antimicrobial peptides MOp2 and MOp3, which were isolated from hydrolysates of Moringa oleifera seeds proteins, on S. aureus biofilm. The results showed that the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration of MOp2 and MOp3 were both 4mg/mL and 8mg/mL, respectively. At 1×MBIC concentration, both MOp2 and MOp3 exhibited biofilm clearance rates of 63.28% and 67.90%, respectively. It was found by fluorescence microscopy that after treatment with MOp2 and MOp3, biofilm adhesion decreased and bacteria numbers reduced. The initial bacterial adhesion rates decreased by 44.19% and 50.77% after treatment with 1×MBIC of MOp2 and MOp3, respectively, and the surface hydrophobicity also decreased. Enzymatic digestion experiments showed that the extracellular proteins (mass fraction 49.01%) were the most abundant component in S. aureus biofilm extracellular polymeric substances, followed by extracellular polysaccharides and extracellular DNA. MOp2 and MOp3 could inhibit the secretion and synthesis of the three components in extracellular polymeric substances. The results of molecular docking showed that MOp2 and MOp3 could both bind to key biofilm formation proteins AgrA, CshA, LuxS and SarA in S. aureus, which might have certain effects on the quorum sensing system of S. aureus and thus inhibit biofilm formation. The study aimed to provide a theoretical basis for the application of Moringa oleifera seed antimicrobial peptides in preservation of food industry.