A bioassay-guided fractionation of the ethyl acetate extract from the twigs of the hybrid poplar 'Neva', Populus nigra L. × Populus deltoides Marsh, led to the isolation of three flavonoids, which were identified by means of spectrometric and physicochemical analysis as 5-hydroxy-7-methoxy-flavone (1), 5,7-dihydoxy-flavone (2) and 5,7-dihydroxy-flavonol (3). These compounds were further screened for their antimicrobial activity against plant pathogens, including three bacteria (Pseudomonas lachrymans, Ralstonia solanacearum and Xanthomonas vesicatoria) and one fungus (Magnaporthe oryzae). Compounds 2 and 3 showed significant antibacterial activity, with minimum inhibitory concentrations (MICs) ranging from 15 to 25 µg mL(-1), and median inhibitory concentrations (IC(50) values) from 4 to 18 µg mL(-1). The results obtained provide promising baseline information for the potential use of the extract and flavonoids from this plant as antimicrobial agents to help control plant diseases.