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Plukenetia huayllabambana is an edible plant traditionally used to cure wounds and various infections. The present work assessed, for the first time, the antibacterial efficacy of solvent fractions from P. huayllabambana fruits. The crude methanol extract was obtained applying ultrasound-assisted extraction, followed by partitioning through successive depletion among solvents of increasing polarity to yield fractions (n-hexane, dichloromethane, ethyl acetate, and n-butanol). The minimal inhibitory concentration (MIC) was determined following antibacterial testing, using the broth microdilution technique against a panel of drug-resistant Gram-negative and Gram-positive bacteria. Possible modes of action of the most active fraction were also investigated. Gas chromatography&ndash, mass spectrometry (GC&ndash, MS) was used to identify phytocompounds that may account for the recorded activities. Methanol, n-hexane (PH-n-Hex), and ethyl acetate extracts inhibited 100% of studied bacteria, with the recorded MIC ranging from 0.125&ndash, 1 mg/mL. PH-n-Hex appeared as the most active partition, exerting a bacteriostatic effect. PH-n-Hex probably acts by interfering with bacterial biofilm formation, proton pumps, and bacterial cell membrane integrity. The GC&ndash, MS analysis of PH-n-Hex led to the identification of 11 potentially bioactive components, including fatty acids, phytosterol, and diterpene alcohol as major ones. P. huayllabambana can be considered as a plant of pharmacological value&mdash, a source of potent anti-infective drug entities.