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The present study focuses on the biological synthesis, characterization, and antibacterial activities of silver nanoparticles (AgNPs) using extracellular extracts of <i>Aspergillus japonicus</i> PJ01.The optimal conditions of the synthesis process were: 10 mL of extracellular extracts, 1 mL of AgNO₃ (0.8 mol/L), 4 mL of NaOH solution (1.5 mol/L), 30 °C, and a reaction time of 1 min. The characterizations of AgNPs were tested by UV-visible spectrophotometry, zeta potential, scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and thermogravimetric (TG) analyses. Fourier transform infrared spectroscopy (FTIR) analysis showed that Ag⁺ was reduced by the extracellular extracts, which consisted chiefly of soluble proteins and reducing sugars. In this work, AgNO₃ concentration played an important role in the physicochemical properties and antibacterial properties of AgNPs. Under the AgNO₃ concentration of 0.2 and 0.8 mol/L, the diameters of AgNPs were 3.8 ± 1.1 and 9.1 ± 2.9 nm, respectively. In addition, smaller-sized AgNPs showed higher antimicrobial properties, and the minimum inhibitory concentration (MIC) values against both <i>E. coli</i> and <i>S. aureus</i> were 0.32 mg/mL.