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Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae) is a medicinal plant traditionally used in Brazil to treat infectious diseases. The extracts obtained of the leaves from P. regnellii were investigated for their antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). The ethyl acetate extract presented a good activity against MRSA, with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 16 μg/mL. Based on this finding, the ethyl acetate extract was fractionated by silica gel column chromatography into nine fractions. The hexane fraction was active against MRSA (MIC at 4 μg/mL). Further column chromatography separation of the hexane fraction afforded the pure compound eupomatenoid-5. The structure of the compound was established by spectral data (1H and 13C NMR HSQC, HMBC, gNOE, IR and MS). Eupomatenoid-5 was the only compound active on the bacterium. The antibacterial property of P. regnellii extract provides preliminary scientific validation for the traditional medicinal use of this plant. The active compound eupomatenoid-5 should be further studied in animal models to verify in vivo efficacy and toxicity.