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A <i>Burkholderia gladioli</i> strain, named BBB-01, was isolated from rice shoots based on the confrontation plate assay activity against several plant pathogenic fungi. The genome of this bacterial strain consists of two circular chromosomes and one plasmid with 8,201,484 base pairs in total. Pangenome analysis of 23 <i>B. gladioli</i> strains suggests that <i>B. gladioli</i> BBB-01 has the closest evolutionary relationship to <i>B. gladioli</i> pv. <i>gladioli</i> and <i>B. gladioli</i> pv. <i>agaricicola</i>. <i>B. gladioli</i> BBB-01 emitted dimethyl disulfide and 2,5-dimethylfuran when it was cultivated in lysogeny broth and potato dextrose broth, respectively. Dimethyl disulfide is a well-known pesticide, while the bioactivity of 2,5-dimethylfuran has not been reported. In this study, the inhibition activity of the vapor of these two compounds was examined against phytopathogenic fungi, including <i>Magnaporthe oryzae</i>, <i>Gibberella fujikuroi</i>, <i>Sarocladium oryzae</i>, <i>Phellinus noxius</i> and <i>Colletotrichum</i><i>fructicola</i>, and human pathogen <i>Candida albicans</i>. In general, 2,5-dimethylfuran is more potent than dimethyl disulfide in suppressing the growth of the tested fungi, suggesting that 2,5-dimethylfuran is a potential fumigant to control plant fungal disease.