Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.