This study was to isolate antibacterial compounds from Gleditsia sinensis Lam. spines through bioassay-guided fractionation (against a Gram-positive bacterium Xanthomonas vesicatoria and a Gram-negative bacterium Bacillus subtilis). The crude ethanol extract of G. sinensis spines was partitioned sequentially with solvents of increasing polarity. The ethyl acetate fraction, which exhibited the most significant antibacterial activities among all the solvent fractions, was further separated by column chromatograph, yielding seven phenolic compounds including ethyl gallate (1) and caffeic acid (7), and five flavonoids, dihydrokaempferol (2), eriodictyol (3), quercetin (4), 3,3',5',5,7-pentahydroflavanone (5) and (-)-epicatechin (6). Compounds 4, 5 and 7 showed moderate inhibitory activities against both bacterial species, with compound 7 having the lowest minimal inhibitory concentration (MIC) of 0.125 mg mL(-1), while compounds 1 and 2 showed a weak inhibitory activity only against B. subtilis (MIC 1.00 mg mL(-1)), and compounds 3 and 6 showed insignificant activity against the two bacteria.