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Citrus canker is an important disease caused by Xanthomonas axonopodis pv. citri that affects citrus species. We isolated a bacterium denominated QC-Y with a strong inhibitory effect on citrus canker from navel orange leaves. The isolate was identified as Bacillus amyloliquefaciens based on the morphological, physiological, and biochemical characteristics and the 16S rDNA sequence analysis. The inhibitory activity of the pathogen was significantly affected by environmental factors such as the medium, inoculation amount, media volume, and pH. The biocontrol strain QC-Y effectively colonised on navel orange leaves, and the colonisation gradually decreased with time. Twelve days after inoculation, the isolate maintained a certain population level in the leaves. Mancozeb demonstrated a strong inhibitory effect on the growth of QC-Y; Chlorpyrifos at high concentrations inhibited QC-Y. Thiophanate-methyl, Bordeaux mixture, Kasugamycin, Imidacloprid, amino acid, Difenoconazole, Etoxazole, Alphacypermethrin, Buprofezin, Spirodiclofen, Avermectin, and Pyraclostrobin had no effect on the growth of QC-Y. In the detached leaf assay, compared with the leaves inoculated with the pathogens only, the disease incidence of the leaves treated with QC-Y was reduced by 77.5% and the lesions were smaller. Our findings reveal that Bacillus amyloliquefaciens QC-Y can be used as a potential biocontrol agent against the citrus canker disease.