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