Bacillus amyloliquefaciens LBM 5006 produces antagonistic activity against pathogenic bacteria and phytopathogenic fungi, including Aspergillus spp., Fusarium spp., and Bipolaris sorokiniana. PCR analysis revealed the presence of ituD, but not sfp genes, coding for iturin and surfactin, respectively. The antimicrobial substance produced by this strain was isolated by ammonium sulfate precipitation, gel filtration chromatography and 1-butanol extraction. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates the presence of peptide bonds and acyl group(s). The antimicrobial substance was resistant to proteolytic enzymes and heat treatment, and was reactive with ninhydrin. Mass spectroscopy analysis indicated that B. amyloliquefaciens LBM 5006 produces two antimicrobial peptides, with main peaks at m/z 1,058 Da and 1,464 Da, corresponding to iturin-like and fengycin-like peptides, respectively. B. amyloliquefaciens LBM 5006 showed significant activity against phytopatogenic fungi, showing potential for use as a biocontrol agent or production of antifungal preparations.