To purify and analyse antimicrobial substances produced by the tomato pathogen Clavibacter michiganensis ssp. michiganensis (Cmm), with potential application in control of Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. After selection of a suitable producer and indicator strain, antimicrobial compounds were isolated using chromatographic techniques. The resulting preparations were analysed with respect to heat and protease sensitivity, amino acid composition, amino acid sequence and mass. Using this procedure we discovered one post-translationally modified 2145 Da peptide bacteriocin, one 14 kDa antimicrobial protein as well as low molecular weight (< 1000 Da) antimicrobial compounds, putatively belonging to the tunicamycin family. Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. This study describes the first attempt to characterize antimicrobial substances from Cmm at the molecular level. This is an important step towards investigation of the possible use of these compounds to control the potato ring rot pathogen.