Manilkara rufula belongs to family Sapotaceae and has received little attention regarding its pharmacological properties and chemical composition. Therefore, our objective was to determine the farmacological activities and preliminary chemical profile of the stem bark from M. rufula. The plant stem bark of M. rufula was collected and ethanolic extract and fractions were prepared. The antioxidant activity was determined by •DPPH and ABTS•+ scavenger methods and inhibition of β-carotene oxidation. The minimum inhibitory and bactericidal concentrations were estimated through the broth microdilution method against gram positive and negative bacteria. Cytotoxicity, antioxidant and cytoprotection potential were investigated on culture macrophages. Antiproliferative activity was evaluated by Alamar Blue method. The in vitro arginase activity from Leishmania amazonensis was determined in presence of the samples. In addition, their effects on survival of L. amazonensis and Trypanosoma cruzi amastigotes in mice macrophages were assessed. The preliminary phytochemical profile was evaluated by qualitative methods of classical phytochemistry, infrared spectroscopy, LC-MS/MS and CG-MS. The samples presented antioxidant, bactericidal, leishmanicidal and trypanocidal properties without toxic characteristics on normal cells. Triterpenes were observed in the hexane fraction, while glycosides, aromatic rings, fatty esters, proanthocyanidin dimers and trimers, catechin and various terpenes were observed in the other fractions.