Deguelia nitidula (Benth.) A.M.G.Azevedo & R.A.Camargo (Fabaceae) is an herbaceous plant distributed in the Brazilian Amazon, and it is called "raiz do sol" (sun roots). On Marajó Island, quilombola communities use its prepared roots to treat skin diseases commonly caused by fungi, viruses, and bacteria. Thus, in this study, the extract, and its fractions from D. nitidula roots were used to perform in vitro cytotoxic and antibacterial assays against Staphylococcus aureus strains. Thereafter, liquid chromatography-mass spectrometry (LC-MS) was used for the metabolite annotation process. The ethanolic extract of D. nitidula roots show significant bactericidal activity against S. aureus with IC50 82 μg.mL-1 and a selectivity index (SI) of 21.35. Furthermore, the SREFr2 and SREFr3 fractions show a potent bactericidal activity, i.e., MIC of 46.8 μg.mL-1 for both, and MBC of 375 and 93.7 μg.mL-1, respectively. As showcased, SREFr3 shows safe and effective antibacterial activity mainly in respect to the excellent selectivity index (SI = 82.06). On the other hand, SREFr2 shows low selectivity (SI = 6.8), which characterizes it as not safe for therapeutic use. Otherwise, due to a limited amount of reference MS2 spectra in public libraries, up to now, it was not possible to perform a complete metabolite annotation. Despite that, our antibacterial results for SREFr3 and correlated substructures of amino acid derivatives show that the roots of D. nitidula are a natural source of specialized metabolites, which can be isolated in the future, and then used as a support for further bio-guided research, as well as natural drug development.