AbstractThis study was carried out to screen a potential probiotic microbe with broad‐spectrum antagonistic activity against food‐borne pathogens and identify the antimicrobial compounds. Based on morphological and molecular analysis, a newBacillusstrain with the ability to produce effective antimicrobial agents was isolated from the breeding soil of earthworms and identified as having a close evolutionary footprint toBacillus amyloliquefaciens. The antimicrobial substances produced byB. amyloliquefaciensshow effective inhibition ofAspergillus flavusandFusarium oxysporumin an agar diffusion assay. Antimicrobial agents were identified as a series of fengycin and its isoforms (fengycin A and fengycin B) after being submitted to RT‐HPLC and MALDI‐TOF MS analyses. To evaluate the probiotic activity of theB. amyloliquefaciens, antibiotic safety and viability of the isolated strain in a simulated gastrointestinal environment were carried out. The safety test result revealed that strain LPB‐18 is susceptible to multiple common antibiotics. Moreover, acidic condition and bile salts assay were carried out, and the results revealed that it couble be a potential probiotic microbeB. amyloliquefaciensLPB‐18 is good choice for biological strains in agricultural commodities and animal feedstuffs.