Clinical samples from 71 bovine from different Brazilian states were processed for the analysis of anaerobe organisms with emphasis on the isolation and characterization of Clostridium spp. From these, eighty-nine Clostridium perfringens strains were recovered: 32 from liver, 19 from intestinal contents, 14 from kidney, 6 from rumen, 5 from nervous system, 4 from bone marrow, 2 from udder tract, blood, spleen and lung, and one from muscle. Four reference Clostridium perfringens types A, B, C, and D were used as controls in this study. All isolates were cultivated in appropriate media, and after centrifugation the supernatant and sediment were separated. From pure supernatant post exopolysaccharide (EPS) extraction, mouse toxigenicity tests were performed, determining protein and protein plus carbohydrate, respectively. ELISA was performed from sediments. The results showed that 51 (57.3%) of the isolates were toxigenic to mice when inoculated by intraperitoneal route; bacteria from different organs had variable patterns of toxigenicity. Toxigenicity of EPS extracts was only expressed when protein concentration was 0.04 mg/mL and between 0.31 and 0.5 mg/mL for carbohydrate. Isolates were characterized as toxigenic when showing optimum protein and carbohydrate concentrations.