Acanthamoeba causes infections in humans and other animals and it is important to develop treatment therapies. Jatropha curcas, Jatropha gossypifolia and Euphorbia milii plant extracts synthesized stable silver nanoparticles (AgNPs) that were relatively stable. Amoebicidal activity of J. gossypifolia, J. curcas and E. milii leaf extracts showed little effect on viability of Acanthamoeba castellanii trophozoites. Plant-synthesized AgNPs showed higher amoebicidal activity. AgNPs synthesized by J. gossypifolia extract were able to kill 74–27% of the trophozoites at concentrations of 25–1.56 μg mL−1. AgNPs were nontoxic at minimum inhibitory concentration with peripheral blood mononuclear cells. These results suggest biologically synthesized nanoparticles as an alternative candidate for treatment of Acanthamoeba infections.