The extracellular biosynthesis of silver nanoparticles (AgNPs) using a bacterial system could be a probable approach, avoiding using destructive chemicals. The aim of this study was the biosynthesis of silver nanoparticles and the comparison of their antibacterial activity against Bacillus cereus and Serratia marcescens. Biosynthesis of AgNPs was done using novel strains of alkalophile Isoptericola variabilis sp.IRSH1 and waste agriculture. AgNPs were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Their synthesis was optimized by experimental design using response surface methodology (RSM). The antibacterial activities of the silver nanoparticles were examined by the standard Kirby–Bauer disc diffusion method on Muller–Hinton agar plates. The results showed the optimized silver nanoparticles were formed with an average size of 77.30 nm and 0.45 polydispersity index (PDI). The antibacterial property of the nanoparticles increased with their increasing concentrations and the maximum inhibition zones against Bacillus cereus and Serratia marcescens were found 9.66 mm and 10.66 mm in 3000 µg/ml concentration of silver nanoparticles, respectively. Despite reducing the density of AgNPs from 3000 µg/ml concentration to 500 and 100 µg/ml concentrations, antibacterial activity against Bacillus cereus and Serratia marcescens were still visible. Consequently, the biosynthesized AgNPs has efficient antibacterial activity against Bacillus cereus and Serratia marcescens. The results indicate that Serratia marcescens is approximately five times more sensitive than Bacillus cereus to silver nanoparticles.