This study investigates the use of Amazon red propolis for the green synthesis of antibacterial silver nanoparticles (AgNPs). A hydroalcoholic propolis extract was fractionated to yield fractions rich in phenolic compounds. High-performance liquid chromatography identified calycosin as a chemical marker, along with liquiritigenin, isoliquiritigenin, and formononetin. The propolis extract and the chloroform fraction inhibited Staphylococcus aureus growth with a bactericidal effect, while for Escherichia coli, a bacteriostatic effect was observed at concentrations below 1000 µg/mL. The AgNPs were characterized by UV-Vis spectroscopy, confirming surface plasmon resonance, and their morphology and size were analyzed using dynamic light scattering and transmission electron microscopy, with elemental composition assessed via energy-dispersive spectroscopy. X-ray diffraction and selected area electron diffraction showed the crystalline structure. Fourier-transform infrared spectra indicated that carboxyl and hydroxyl groups in flavonoids reduce silver ions and contribute to the nanoparticles' chemical stability. The nanoparticles synthesized from the propolis extract showed enhanced antibacterial potential, with efficacy against E. coli (MIC 25 µg/mL, MBC 50 µg/mL) compared to S. aureus (MIC 100 µg/mL, MBC 100 µg/mL), and demonstrated selectivity for Gram-negative bacteria over murine macrophages (CC 83.92 μg/mL). These nanoparticles exhibited maximum absorption in the UV-Vis region at 418 nm, with a spherical silver core encapsulated by compounds from propolis, hydrodynamic diameter of 252.47 ± 1.42 nm, a zeta potential of -51.80 ± 0.70 mV, and a polydispersity index of 0.218 ± 0.01, indicating high chemical stability. These findings underscore Amazon red propolis as a sustainable resource for developing selective and stable antibacterial agents.