In this study, Retama monosperma extract (RME) was used for the green synthesis of silver nanoparticles (RME-AgNPs). RME's phenolic profile was identified by liquid chromatography coupled to mass spectroscopy (LC-ESI/MS/MS) technique. A tentative identification of 21 phenolic metabolites from the extract was performed. The produced RME-AgNPs showed UV absorbance at 443 nm. FTIR spectroscopy confirmed the presence of RME functional groups. In addition, XRD analysis confirmed the crystallography of RME-AgNPs via exhibiting peaks with 2θ values at 38.34°, 44.29°, and 64.65°. RME-AgNPs were spherical with particle sizes ranging from 9.87 to 21.16 nm, as determined by SEM and HR-TEM techniques. The zeta potential determined the particle's charge value as -15.25 mv. RME-AgNPs exhibited significantly higher antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) compared to RME. Moreover, the SEM images of green-synthesized nanoparticles revealed severe damage and deformation in the bacterial cell wall of the different strains subjected to the current investigation. The bioinformatics study identified 266 targets, among which only 41 targets were associated with bacterial infections. The PI3K-Akt and Relaxin signaling pathways were the top KEGG signaling pathways. Molecular docking was also performed for the 21 identified compounds at the TNF-α active site; kaempferol-3-O-robinoside-7-O-rhamnoside had a higher binding energy (-6.8084). The findings of this study warrant the use of green-synthesized AgNPs from Retama monosperma as potential antibacterial agents.