Some Acanthamoeba strains are able to cause Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide because of their pathogenicity. The treatment of Acanthamoeba infections is complicated due to the existence of a highly resistant cyst stage in their life cycle. Therefore, the elucidation of novel sources of anti-Acanthamoeba agents is an urgent need. In the present study, an evaluation of the antioxidant and anti-Acanthamoeba activity of compounds in flower extracts of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile methanol extract was the most active showing an IC50 of 66.235 ± 0.390 mg/ml, low toxicity levels when checked in murine macrophage toxicity model and presented also antioxidant properties. Moreover, a bio-guided fractionation of this extract was developed and led to the identification of a mixture of coumarins as the most active fraction. These results suggest a novel source of anti-Acanthamoeba compounds for the development of novel therapeutic agents against Acanthamoeba infections.