Background: Biofilm formation activity of pathogenic bacteria plays an important role in the pathogenesis and progression of various diseases caused by bacterial infections. It has been reported that lupinifolin, a major phytochemical isolated from Derris reticulata stem, possesses an antibacterial activity against Streptococcus mutans and Staphylococcus aureus. Nonetheless, its actions on biofilm formation properties of S. mutans and S. aureus have not been clearly established. ?Objectives: This study aimed to investigate the antibacterial and antibiofilm formation activities of lupinifolin derived from D. reticulata stem against S. mutans and S. aureus. Subjects and Methods: The minimum inhibitory concentration (MIC) was evaluated using the microbroth dilution method. The antibiofilm formation activity of lupinifolin was conducted at various incubation periods using the crystal violet biofilm formation assay. Results: The MICs of lupinifolin against S. mutans and S. aureus were 4 and 8 μg/mL, respectively. Lupinifolin at the concentrations of sub-MICs had significant inhibitory actions against both sucrose-dependent and sucrose-independent biofilm formations of S. mutans. The lowest median inhibitory concentrations (IC50s) were found at the incubation periods of 12 h (0.57 ± 0.08 μg/mL) and 20 h (0.21 ± 0.04 μg/mL) against sucrose-dependent and sucrose-independent S. mutans biofilm formations, respectively. In addition, at its sub-MICs, lupinifolin also produced a significant inhibition against S. aureus biofilm formation with the lowest IC50 of 0.22 ± 0.03 μg/mL observed at 6-h incubation. Conclusion: These results evidently indicated that lupinifolin can potentially be developed further as a natural product-derived antibiofilm-forming agent for the prevention and/or treatment of biofilm-associated bacterial infections.