Sorindeia nitidula is used by traditional practitioners to treat influenza illnesses with cephalgia and febrile aches. However, the potential active ingredients for its remarkable antioxidant, anti-HIV and antitrypanosomal activities remain unexplored. The present study aims to evaluate the antioxidant, anti-HIV and antitrypanosomal activities of the ethyl acetate extract of S. nitidula (SN) in order to screen out the bioactive compounds and to analyze their possible mechanisms of action. Overall, 21 phenolic compounds were annotated, by using the MS and MS/MS information provided by the QTOF-MS. In vitro assays on the extract revealed potent antioxidant (IC50 = 0.0129 mg/mL), anti-HIV (IC50 = 1.736 mg/mL), antitrypanosomal (IC50 = 1.040 µM) activities. Furthermore, SN did not present cytotoxic effect on HeLa cancer cell lines (IC50 = 0.045 µM). Molecular docking revealed that the potential ligands exhibited strong binding ability and inhibitory activities on trypanosome. The integrated strategy based on LC-ESI-QTOF-MS/MS and molecular docking provided a powerful tool and a multidimensional perspective for further exploration of active ingredients in S. nitidula responsible for the antioxidant, anti-HIV and antitrypanosomal activities.