This work aimed to characterize antagonistic bacteria from the field-grown barley rhizosphere, and evaluate their potential for growth promotion and biocontrol of Fusarium wilt on watermelon caused by Fusarium oxysporum f. sp. Niveum (FON). Seven bacteria were isolated and screened for plant growth promoting and antagonistic traits. Based on the results of phenotypic characterization and 16S rRNA gene sequencing, the isolates were identified to be related to Bacillus methylotrophicus (DMK-1), Bacillus amyloliquefaciens subsp. plantarum (DMK-7-2), Bacillus cereus (DMK-12), Pseudomonas brassicacearum subsp. brassicacearum (DMK-2), Pseudomonas veronii (DMK-3), Paenibacillus polymyxa (DMK-8), and Ensifer adhaerens (DMK-17). All the isolates were positive for the production of indole-3-acetic acid (IAA) and ammonia (NH 3), while negative for the production of hydrogen cyanide (HCN). Six bacteria strains (except DMK-17) were able to phosphate solubilization. All the bacteria strains, except DMK-8, were able to produce iron siderophore complexes, and possessed the proteolytic activity. Greenhouse experiment indicated six strains can decrease diseased percentage caused by FON. All the isolates enhanced plant biomass, six strains increased root volume, six strains increased root system activity in greenhouse test. Inoculation of mixtures of seven plant growth promoting rhizobacteria could be more effective in plant growth promotion and biocontrol of Fusarium wilt in watermelon.