Bacterial leaf blight caused by Xanthomonas axonopodis is a major problem in the production of eucalyptus seedlings. The pathogen causes leaf lesions that reduce the photosynthetic area and limit plant growth. The objective of this study was to evaluate the antibiotic activity of secondary metabolic compounds of a Pseudomonas sp. against X. axonopodis, following extraction with dichloromethane and fractionation by vacuum liquid chromatography. The antibiotic activity of an ethyl acetate phase (F3 fraction) was determined by agar diffusion tests and bioautography. The minimum inhibitory concentration of F3 fraction and the action of F3 on the cell morphology of X. axonopodis was determined by scanning electron microscopy. Electron microscopy revealed changes in exopolysaccharides and morphology of treated cells. When eucalyptus seedlings were sprayed with the F3 fraction before or after inoculation with X. axonopodis in a greenhouse experiment the number of leaf lesions was respectively 93.9% and 89.7% lower than in untreated control plants. High antibiotic activity of the F3 fraction against X. axonopodis and leaf blight suggests that it has potential to control the disease in eucalyptus seedlings.