[Objective] To screen effective biocontrol agents from healthy banana rhizosphere in a diseased field and further study the inhibition mechanism. [Methods] Double-deck plates and fermentation antagonism study were used as primary- and secondary-screening for antagonists. Observing strains were identified based on physiological and biochemical tests, 16S rRNA gene sequencing and specific gene amplification. Crude extracts of fermentation were observed by using acid precipitate method and then added to the fermentation of pathogen for 5 days. Concentration of protein, malondialdehyde (MDA), ergosterol and pectinase activities were analyzed based on colorimetric method and HPLC to find out the effect of antagonists crude extracts on the growth of pathogen. [Results] Two antagonists, named as H-2 and H-7, were observed and identified as Bacillus subtilis (GenBank: KX791428) and Bacillus amyloliquefaciens (GenBank: KX791430), respectively. Fusarium wilt of banana was suppressed by these two strains and the biocontrol efficacies were 59.1% and 53.0%, respectively, based on the greenhouse condition. Concentration of MDA was significantly increased to 0.55 μmol/L and 0.48 μmol/L by treating the pathogen hyphae with suspension of H-2 and H-7, respectively. Meanwhile, concentration of protein, ergosterol and pectinase activities were significantly decreased in both H-2 and H-7 treatments and the lower index values were observed by H-2 by showing as 0.15 mg/g, 1.31 mg/g and 0.008 7 U/mL, respectively, which were significantly lower than those of CK (0.25 mg/g, 1.96 mg/g and 0.035 U/mL). [Conclusion] In conclusion, two antagonists screened from healthy banana rhizosphere soil were able to inhibit the growth of pathogen by increasing the lipid peroxidation of pathogen hyphae and decreasing the synthesis of metabolism products, which provide theoretical basis on the biocontrol application of two antagonists.