Four alkaloids (Sanguinarine, 6-Methoxyl-dihydro-chelerythrine, Cryptopine and β-Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay-guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram-negative strains than other three alkaloids, with MIC values of 12.5 mg L−1 for A. hydrophila and 50 mg L−1 to other pathogenic bacteria. Followed by 6-methoxyl-dihydro-chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L−1 for A. hydrophila, 100 mg L−1 for V. harveyi, and 125 mg L−1 for both V. anguillarum and A. salmonicida. Cryptopine and β-allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L−1 for A. hydrophila and 200 mg L−1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.