The antibacterial activities of crude extracts from the fruit capsules, leaves, and roots of Macleaya cordata, as well as four isoquinoline alkaloids, including sanguinarine(SAN), chelerythrine(CHE), protopine(PRO), and allocryptopine(ALL) against Xanthomonas citri subsp. citri(Xcc) were evaluated. Combined with determinations of minimum inhibitory concentration(MIC), half maximal inhibitory concentration(IC50), scanning electron microscopy (SEM) observation, and in vivo inoculation assays, the key active components in M. cordata responsible for Xcc inhibition and their effects were identified. The results showed that the crude extract from M. cordata fruit capsules exhibited the most significant antibacterial activity, and its SAN content was significantly higher than those detected in the leaves and roots, as well as other alkaloids in the fruit capsules. SAN and CHE were identified as the key antibacterial components, with MIC values determined to be 4 μg/mL and 8 μg/mL, and IC50 values of (1.723±0.002) μg/mL and (6.164 ±0.106) μg/mL, respectively. Results from scanning electron microscopy demonstrated that 4 μg/mL SAN induced morphological deformation and surface depression in Xcc after 4 h co-culture, while 8 μg/mL CHE compromised bacterial membrane integrity, and the integrity of the bacterial membrane structure was damaged, indicating that SAN and CHE had a strong inhibitory effect on Xcc at very low doses. Results from in vivo inoculation assays of Xcc showed that, under the condition of inoculation followed by spraying, the optimal antibacterial effects were achieved by SAN at 30 μg/mL and CHE at 10-20 μg/mL; under the condition of spraying followed by inoculation, the optimal mass concentrations were determined to be 20 μg/mL for SAN, and 30 μg/mL and 50 μg/mL for CHE. The best inhibitory effect against Xcc was exhibited by the SAN∶CHE combination at a ratio of 2∶1(30 μg/mL∶15 μg/mL).