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In this paper, the virulence of Bordeaux mixture against Alternaria alternata was determined, and the effects of Bordeaux mixture on phyllosphere microbial community structure and metabolic function of healthy and diseased tobacco leaves were determined by high-throughput sequencing and Biolog metabolic phenotype technology. Results showed that Bordeaux mixture had a weak inhibitory activity against A. alternata with EC50 values for inhibiting mycelial growth and spore germination of 450.19 and 757.17 mg/L, respectively. The phyllosphere bacteria in both healthy and diseased tobacco were distributed in Proteobacteria (6.93% and 39.07%) and Firmicutes (16.45% and 0.65%), and the dominant bacterial genera were Kosakonia (3.46% and 22.38%) and Pseudomonas (0.22% and 5.95%). The phyllosphere fungi in both healthy and diseased tobacco were distributed in Ascomycota (63.82% and 93.74%) and Basidiomycota (6.82% and 2.53%), and the dominant fungal genera were Alternaria (36.48% and 84.52%), Symmetrospora (5.56% and 2.27%) and Cladosporium (14.87% and 6.66%). Bordeaux mixture at 1 500 g/hm² affected the community structure and metabolic function of phyllosphere bacteria and fungi in healthy and diseased tobacco. In the bacterial and fungal communities, the relative abundances of Kosakonia, Sphinomonas, and Lactobacillus decreased, while the relative abundances of 6 bacterial genera including Pseudomonas, Ralstonia increased after 5 days of treatment. The relative abundance of 6 fungal genera including Alternaria, Symmetrospora decreased, while the relative abundance of 10 fungal genera including Didymella, Metarhizium increased after 5 days of treatment. The effects of Bordeaux mixture on phyllosphere fungi and bacteria gradually decreased after 10 and 15 days of treatment. Twenty-nine carbon sources including carbohydrates, amino acids, carboxylic acids, amphiphiles, polymers, and amine/amino compounds could be efficiently metabolized by phyllosphere microorganisms of healthy and diseased tobacco leaves, but the metabolism of α-butyric acid was weak. The metabolic inhibition activity of Bordeaux mixture on phyllosphere microorganisms decreased gradually with the increase of time. The results of the study revealed the regularity of the effect of Bordeaux mixture on the microbes of tobacco leaves after different periods of application and provided a reference for understanding the ecological benefits of the fungicide in the effective period. [ABSTRACT FROM AUTHOR]