In chronic infections caused by Staphylococcus aureus, biofilm is a major virulence factor. In Staphylococcus aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances and are highly tolerant to antimicrobial drugs. However, the lack of effective solutions to inhibit biofilm formation remains a challenge, and the mechanism of inhibition of biofilm formation targeting extracellular polymeric substances is unclear. The aim of the present study was to investigate the inhibitory mechanisms of Plumbagin against Staphylococcus aureus biofilms formation by affecting secretion of extracellular polymeric substances using the high-content screening. Our results showed Plumbagin (16 µg/mL) inhibited biofilm formation, revealing a significant reduction in both biomass and bacterial metabolic activity, and disrupted the biofilm structure, leading to a significant decrease in both biological volume and average thickness (P ≤ 0.01). High-content screening imaging indicated that the Plumbagin treatment induced alterations in the extracellular polymeric substances of Staphylococcus aureus biofilm, significantly reducing the quantities of extracellular polysaccharide, proteins and extracellular DNA. Interestingly, extracellular DNA within the matrix was found to be the most sensitive to Plumbagin treatment. Extracellular DNA formation was significantly inhibited at a concentration of 4 µg/mL, whereas the inhibition of extracellular polysaccharide and proteins required a higher concentration of 8 µg/mL. Overall, these results demonstrated the inhibitory effects of Plumbagin on Staphylococcus aureus biofilm formation and extracellular polymeric substances secretion, suggesting that extracellular DNA may be a potential target for the anti-biofilm activity of Plumbagin. These findings will provide new insights into the mode of action of Plumbagin in treating infections caused by Staphylococcus aureus biofilms.