The utilize of microorganisms in the biosynthesis of nanoparticles from metals ions appear as an eco-friendly approach and alternative to the harmful traditional approaches in the environment. In this study, we synthesized silver nanoparticles (AgNPs) using Serratia marcescens subsp. sakuensis supernatant. The optimization factors of the production silver nanoparticles were carried out by seven parameters (medium growth, incubation time, the ratio of mixing culture supernatant to silver nitrate, temperature degree, pH level, silver nitrate concentration and agitation speed). The optimum silver nanoparticles production was achieved at pH 7, AgNO3 concentration 2mM, temperature 30ºC, mixing ratio of silver nitrate to volume of culture supernatant 20:50 ml, incubation time 48h and agitation speed 140 rpm for AgNPs production. Also, the synthesized silver nanoparticles were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM), powder X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FTIR) and Energy-dispersive X-ray spectroscopy (EDX). The surface plasmon absorbance spectra of AgNPs was observed at 430 nm, and transmission electron microscopy images showed that the diameter of well-dispersed AgNP (10–20 nm). In addition, the antibacterial activity was studied and the obtained results of the synthesized AgNPs by Serratia marcescens subsp. sakuensis showed a good antibacterial activity against the studied pathogenic bacteria.