Objective: To evaluate the in vitro anti-Trypanosoma cruzi (T. cruzi) activity of organic extracts prepared from halophyte species collected in the southern coast of Portugal (Algarve), and chemically characterize the most active samples. Methods: Acetone, dichloromethane and methanol extracts were prepared from 31 halophyte species and tested in vitro against trypomastigotes and intracellular amastigotes of the Tulahuen strain of T. cruzi. The most active extract was fractionated by preparative HPLC-DAD, affording 11 fractions. The most selective fraction was fully characterized by 1H NMR. Results: From 94 samples tested, one was active, namely the root dichloromethane extract of Juncus acutus (IC50 < 20 μg/mL). This extract was fractionated by HPLC, affording 11 fractions, one of them containing only a pure compound (juncunol), and tested for anti-parasitic activity. Fraction 8 (IC50 = 4.1 μg/mL) was the most active, and was further characterized by 1H NMR. The major compounds were phenanthrenes, 9,10-dihydrophenanthrenes and benzocoumarins. Conclusion: Our results suggest that the compounds identified in fraction 8 are likely responsible for the observed anti parasitic activity. Further research is in progress aiming to isolate and identify the specific active molecules. To the best of our knowledge, this is the first report on the in vitro anti T. cruzi activity of halophyte species.