The aim of this work was biochemical characterization and classification of proteinases in pathogenic and non-pathogenic strains of Acanthamoeba spp. The authors showed two proteinase (acid 35 kDa and alkaline 65 kDa) which could be separated electrophoretically. Acid proteinase was inhibited by serine proteinase inhibitors such as DIFP. The second enzyme which was active at alkaline pH, was enhanced by EDTA and inhibited by iodoacetate (IAA) and (p-CMB) p-chloromercuribenzoate. These substances are known to inhibit cysteine type proteinases. The alkaline proteinase was more distinctively active in pathogenic strains and belongs to cysteine class (EC 3.4.22), whereas the acid proteinase was similar active in pathogenic and non-pathogenic strains and belong to serine class (EC 3.4.21).