Abstract: The aims of this study were to identify two Bacillus spp., CYY-6 and CYY-42, isolated from the rhizosphere of alfalfa, and to evaluate their ability to inhibit Fusarium oxysporum, the causal pathogen of alfalfa root rot. Based on physiological, biochemical, morphological, and 16S rDNA sequencing analyses, strain CYY-6 was identified as Bacillus licheniformis and CYY-42 as Brevibacillus brevis. Both strains were able to synthesize siderophores, although at different levels. In a dual culture assay, both strains inhibited the growth of F. oxysporum. Based on the inhibition zones, the inhibitory rates of CYY-6 and CYY-42 against F. oxysporum were 49.3% and 56.3%, respectively. The fermentation broth of each strain resulted in deformed growth of the mycelia and spores of F. oxysporum. The mycelia were twisted, broken, or fragmented, and the number of spores was significantly decreased. Strains CYY-6 and CYY-42 showed 57.55% and 64.03% inhibition of alfalfa root rot in pot experiments, indicating that both strains have potential applications in controlling alfalfa root rot.