Bacterial diseases of onion (Allium cepa L.) are serious problems in Poland. In this study bacterial strains were isolated from onion bulbs with soft-rot symptoms. Pathogenicity tests were conducted on onion bulbs and also on tobacco leaves on whose basis twelve isolates were chosen to further characterization. These isolates were identified using physiological and biochemical tests, and confirmed by species-specific PCR, ERIC-PCR and sequence analysis of 16S rRNA, gyrB, lepA, phaC, recA gene fragments. All examined isolates were identified as Burkholderia gladioli. It is the first report of the occurrence of B. gladioli on onion as the cause of onion disease in Poland.