This study evaluated in vitro activity of ethanol extract, fractions, and isolated substance from Amazon species against promastigotes of L. amazonensis. The ethanol extracts were concentrated and fractionation. The anti-promastigote activity was evaluated through the cell viability assessment method (MTT). The ethanol extract, fractions, and isolated substance from Himatanthus articulatus and Parahancornia fasciculata were inactive in promastigote of L. amazonensis, as the ethanol extract of Physalis angulata. The hexane fractions from different parts of Montrichardia linifera showed anti-promastigote activity probably due to the presence of steroids and terpenes. All species in studies were inactive, except of M. linifera. The few polar constituents can be responsible for the activity. Therefore, the isolation and purification of the active on L. amazonensis promastigotes are urgently required.