This study evaluated the effect of derriobtusone A, a flavonoid isolated fromLonchocarpus obtusus, on two important pathogenic bacteria,Staphylococcus aureusandEscherichia coli, as well as its antioxidant activity and toxicity. Planktonic growth assays were performed, and the inhibition of biofilm formation was evaluated. In addition, antioxidant activity was assessed by DPPH radical scavenging assay, ferrous ion chelating assay, ferric-reducing antioxidant power assay, andβ-carotene bleaching assay. Toxicity was evaluated by the brine shrimp lethality test. Results showed that derriobtusone A completely inhibited the planktonic growth ofS. aureusat 250 and 500 μg/mL; however, it did not have the same activity onE. coli. Derriobtusone A reduced the biomass and colony-forming unit (cfu) ofS. aureusbiofilm at concentrations of 250 and 500 μg/mL. In various concentrations, it reduced the biofilm biomass ofE. coli, and, in all concentrations, it weakly reduced the cfu. Derriobtusone A showed highly efficient antioxidant ability in scavenging DPPH radical and inhibitingβ-carotene oxidation. The compound showed no lethality toArtemiasp. nauplii. In conclusion, derriobtusone A may be an effective molecule againstS. aureusand its biofilm, as well as a potential antioxidant compound with no toxicity.