Acanthamoeba, a free-living protozoan widely distributed in the environment, can cause Acanthamoeba keratitis, a significant ocular microbial infection. The illness can result in blindness when not properly treated in the initial stage. Plants of the genus Piper (Piperaceae) are used in folk medicine for the antibacterial, antifungal and antiprotozoan properties. In this work, the chemical composition and the amoebicidal activity of Piper hispidinervum essential oil were investigated. The leaves of the fresh plant submitted to steam distillation yielded 0.95% (w/w) of essential oil that was analyzed by gas chromatography–mass spectrometry (GC/MS) being safrole the main component, representing 85.08% of the oil. For the assessment of the amoebicidal activity concentrations of 0.5, 0.250, 0.125 and 0.0625 mg/mL were tested. The essential oil, at the concentrations of 0.5 mg/mL, was lethal to 100% of the A. polyphaga trophozoites. By the MTT assay it was verified that the essential oil was not cytotoxic to the mammalian cells until the concentration of 0.25 mg/mL. Nevertheless, further studies are necessary in order to verify its applicability in Acanthamoeba keratitis treatment.