The aim of this study was to characterize the biochemical composition of two main IB‐16 Bacillus thuringiensis proteins and to determine their toxicity on the blood‐feeder nematode, Haemonchus contortus. The soluble toxin of IB‐16 strain of B. thuringiensis produces five main bands of proteins, the chemical composition of which might play an important role on the lethal activity. Two bands of proteins around 25 and 70 kDa were chosen and purified by HPLC using a hydrogel column and sephadex‐beads G‐50. Biochemical analysis was carried out to determine enzyme and carbohydrate moieties on purified fractions of the 25 and 70 kDa proteins. In addition, in vitro assays were carried out using H. contortus histiotropic larvae (L4) and the purified fractions. Biochemical results showed only enzyme activity in the purified fraction of the 25 kDa protein using gelatine as the substrate. In contrast, carbohydrate moieties were only observed in the purified fraction of the 70 kDa protein. Moreover, IB‐16 B. thuringiensis purified fractions of 70 and 25 kDa showed lethal activity of 67.1% and 17.3% of toxicity on H. contortus L4, respectively.