In this study, we investigated the chemical composition, and antioxidant and antibacterial properties of ethanolic extracts of propolis (EEP) from Melipona quadrifasciata quadrifasciata and Tetragonisca angustula. Chemical composition of EEP was determined by colorimetry and chromatographic (HPLC-DAD and UPLC-Q/TOF-MS/MS) analysis. Antimicrobial activity of EEP was evaluated against gram-positive (S. aureus, methicillin-resistant S. aureus, E. faecalis) and gram-negative (E. coli and K. pneumoniae) bacteria by the minimal inhibitory concentration (MIC) test using the microdilution method. Furthermore, the growth curve and integrity of cell membrane of S. aureus and E. coli were investigated using standard microbiological methods. HPLC-DAD analysis showed that the EEP of M. quadrifasciata quadrifasciata has a more complex chemical composition than the EEP of T. angustula. Moreover, UPLC-MS analyses of M. quadrifasciata quadrifascita indicated flavonoids and terpenes as major constituents. The bactericidal activity of both EEPs was higher against gram-positive bacteria than for gram-negative bacteria. The EEP from M. quadrifasciata quadrifasciata presented MIC values lower than the EEP from T. angustula for all tested bacteria. The EEP from M. quadrifasciata quadrifasciata caused lysis of the bacterial wall and release of intracellular components from both E. coli and S. aureus. Our findings indicate that the chemical composition of propolis from stingless bees is complex and depends on the species. The extract from M. quadrifasciata quadrifascita was more effective against gram-positive than gram-negative strains, especially against S. aureus and methicillin-resistant S. aureus compared to T. angustula extract, by a mechanism that involves disturbance of the bacterial cell membrane integrity.