Propolis has a great diversity in its composition due to numerous factors; therefore, each study is an important contribution to the knoFwledge of its composition and biological action. The objective of this study was to determine the chemical profile and biological activity of propolis produced by Scaptotrigona depilis.Extracts with 70% ethanol (EPE70) and with cereal alcohol (CAPE) were elaborated, and then characterized using UHPLC-ESI(+)-MS/MS. Volatile compounds were extracted and then characterized using gas chromatography mass spectrometry (GC-MS). In addition, antimicrobial activities were verified against resistant strains.The volatile compounds of propolis predominantly consist of sesquiterpenes. Using the exploratory metabolomic approach, compounds of different classes were putatively identified in the ethanolic extracts, of which the most representative were terpenes, and some of the sesquiterpenes identified among the volatiles were also detected. The extracts were shown to be active against Escherichia coli and Staphylococcus aureus bacteria with a minimum inhibitory concentration (MIC) of 0.5 and 1.0 mg mL , respectively.The molecular network approach proved to be determining the chemical profile of S. depilis propolis rapidly and accurately, and led to the identification of lipophilic compounds. The identification of compounds using GC-MS and UHPLC-ESI(+)-MS/MS is complementary and useful for the characterization of propolis.