Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K(I) of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver-Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C' in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.