Abstract: Green-synthesized nanomaterials are anticipated to be extensively applied to inhibit sulfate-reducing bacteria (SRB) in oilfields. Using Bacillus subtilis (B. subtilis) suspension as the raw material and zinc acetate dihydrate (C4H6O4Zn·2H2O) along with silver nitrate (AgNO3) as precursors, Ag-ZnO nanoparticles (NPs) were synthesized using a simple and environment-friendly approach. The resulting Ag-ZnO NPs showed a rough surface morphology combined with high dispersibility, showing a particle size distribution of 33–35 nm. Protease and lipopeptides were identified as potential key agents driving the synthesis of Ag-ZnO NPs. At 40 μg/mL, the Ag-ZnO nano-solution effectively inhibited SRB cell growth and reduced the corrosion rate of carbon steel materials induced by SRB. Notably, reactive oxygen species (ROS) and malondialdehyde (MDA) levels increased significantly in SRB cells treated with Ag-ZnO NPs, while cell viability declined, indicating that the nanoparticles caused irreversible damage to SRB biofilms. The inhibitory mechanisms of the NPs against SRB cells were proposed to involve physical electrostatic interactions, metal ion toxicity, cell membrane disruption, and cell lysis.