The production of antagonistic substance by bacterium present in the infected root canal system (RCS) probably is an important ecological factor for its successful colonization of the local. The objective of this study was to partially characterize an antagonistic substance produced by a Clostridium butyricum isolated from infected RCS. Production of inhibitory compound was evaluated by the agar double layer diffusion technique using Fusobacterium nucleatum and Bifidobacterium adolescentis as indicator bacteria. The physicochemical and biochemical factors tested for the partial characterization were influence of pH and temperature and susceptibility to the action of some proteolytic enzymes. An inhibition zone was observed against the two indicator strains and acidity and bacteriophage were rejected as responsible for this phenomenon. The inhibitory activity showed to be decreasing in a pH range from 3.5 to 6.5 and being stable at temperatures of 60°, 70° and 100°C, but completely inactivated when exposed at 121°C. The antagonistic activity was resistant to the proteolytic action of trypsin, α-chymotrypsin and papain. An antagonistic substance was produced by C. butyricum, which was thermo-resistant and probably of non-protein nature.