Haemophilus parasuis is the pathogen of Gl&aumlsser's disease, a major illness affecting young pigs. The aim of this work was to investigate the antagonistic activity of antimicrobial substances produced by Bacillus spp. against H. parasuis. Among the tested strains, only Bacillus subtilis ATCC 6633 inhibited H. parasuis growth. The antibacterial substance was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-50 and ion exchange chromatography on DEAE-cellulose. The purification was about 100-fold with a yield of 0.33%. The purified substance was resistant up to 80ºC and pH ranging 3 to 7, but the substance lost its activity when it was treated with proteases. The peptide showed a molecular mass of 1083 Da and its sequence was determined by mass spectrometry as NRWCFAGDD, which showed no homology with other known antimicrobial peptides. The complete inhibition of H. parasuis growth was observed at 20 ug ml-1 after 20 min of exposition. The peptide obtained by chemical synthesis also showed antimicrobial activity on H. parasuis. The identification of antimicrobial substances that can be effective against H. parasuis is very relevant to combat a pathogen that causes important lose in swine production.