Pythium insidiosum is an oomycete classified in the kingdom Stramenopila. P. insidiosum hyphae are not able to initiate infection without the secretion of hydrolytic enzymes, which are considered an important factor in microbial virulence.To evaluate the extracellular enzymatic activity of 14 Brazilian P. insidiosum isolates and a standard strain (ATCC 58637) by the API-ZYM System screening method.Zoospores were grown in RPMI 1640 broth, and 65 μL of the liquid phase were inoculated in each cupule of the API-ZYM strips.Differences in the enzymatic activities were observed among the isolates, although phosphohydrolases and ester hydrolases were conspicuous among all isolates. β-glucosidase was also present in most of the isolates. Enzymatic activities of α-glucosidase and chymotrypsin were not observed, differing from a previous study involving Australian isolates and intracellular enzymes.The discrepancy in the enzymatic profile observed among Brazilian P. insidiosum isolates reflects the phenotypic variations found in susceptibility tests.