Five new anti-trypanosomal macrolides, actinoallolides A-E (1-5), were discovered from the cultured broth of Actinoallomurus fulvus MK10-036. The structures of the actinoallolides, including absolute stereochemistry, were elucidated by a combination of spectroscopic analyses and a series of chemical derivatization studies. Actinoallolide A showed the most potent and selective in vitro anti-trypanosomal activity without cytotoxicity. A new class of promising lead compounds was identified for the development of drugs for both sleeping sickness and Chagas disease.