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Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern representing about 60% of S. aureus isolated from hospitalized patients in countries such as USA and Brazil in the last years. Additionally, the ability to adhere to surfaces and the development of biofilms are important properties of pathogenic bacteria involved in medical device-associated infections, and staphylococci are recognized as the major etiologic agents in these situations. The aim of this study is to evaluate three Brosimum acutifolium flavonoids, 4′-hydroxy-7,8(2″,2″-dimethylpyran)flavan (1), brosimine b (2) and 4-hydroxy-lonchocarpin (3), regarding their antibiofilm, antibacterial and antioxidant activities. Flavonoids 1 and 2 were able to reduce S. aureus viability within preformed biofilms in 73% at 50 μM while 2 also reduced biofilm biomass in 48% at 100 μM. Flavonoid 3 was not able to reduce biofilm biomass at assessed concentrations. When tested against methicillin-resistant S. aureus (MRSA) strains, 2 (100 μM) reduced 70%–98% of viable bacteria within 24h-old biofilms. The minimum inhibitory concentration against the methicillin-sensitive Staphylococcus aureus ATCC 25904 was 50 μM for the three compounds. In preliminary assays to evaluate cytotoxicity, 1 was highly hemolytic at concentrations above 50 μM while 2 and 3 did not cause significant hemolysis at 100 μM. The antioxidant activity was observed only in the ethanolic extract and 2. In vivo toxicity evaluations using Galleria mellonella larvae as alternative host model resulted in 83.3% survival for treatment with 1 , 76.7% for 2 , and 100% for 3 at 500 mg/kg. This study highlights the potential of these flavonoids, especially 2 , as antibiofilm agent to control preformed S. aureus biofilms. Image 1 • The flavonoid Brosimine b displayed antibacterial activity against S. aureus (MSSA and MRSA). • Brosimine b reduced bacterial viability within S. aureus preformed biofilms. • Brosimine b presented low toxicity to erythrocytes and G. mellonella larvae. [ABSTRACT FROM AUTHOR]