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Chagas disease, sleeping sickness and malaria are infectious diseases caused by protozoan parasites that kill millions of people worldwide. Here, we performed in vitro assays of <i>Pa-MAP</i>, <i>Pa-MAP1.9</i>, and <i>Pa-MAP2</i> synthetic polyalanine peptides derived from the polar fish <i>Pleuronectes americanus</i> toward <i>Trypanosoma cruzi</i>, <i>T. brucei gambiense</i> and <i>Plasmodium falciparum</i> activities. We demonstrated that the peptides <i>Pa-MAP1.9</i> and <i>Pa-MAP2</i> were effective to inhibit <i>T. brucei</i> growth. In addition, structural analyses using molecular dynamics (MD) studies showed that <i>Pa-MAP2</i> penetrates deeper into the membrane and interacts more with phospholipids than <i>Pa-MAP1.9</i>, corroborating the previous in vitro results showing that <i>Pa-MAP1.9</i> acts within the cell, while <i>Pa-MAP2</i> acts via membrane lysis. In conclusion, polyalanine <i>Pa-MAP1.9</i> and <i>Pa-MAP2</i> presented activity against bloodstream forms of <i>T. b. gambiense</i>, thus encouraging further studies on the application of these peptides as a treatment for sleeping sickness.