The biosynthesis of silver nanoparticles (Ag-NPs) is a new methodology in nanotechnology with a hopeful implementation in medicine, food control, and pharmacy. The objective of the present research was to conduct a green synthesis of Ag-NPs using the cell-free supernatant of Lactobacillus plantarum and Lactobacillus brevis and evaluate their antibacterial and antifungal activities. The production of Ag-NPs was confirmed by the color alteration from yellow to brown. Using the UV-visible spectrophotometer, the biosynthesized Ag-NPs indicated an absorption peak at 410 nm. The transmission electron microscope was used for the determination of the size and morphology of the nanoparticles. Nanoparticles appeared in spherical or polyhedral form, poly-dispersed and their diameter ranged from 5 to 40 nm. The X-ray diffraction analysis exhibited the crystalline nature of the particles with a face-centered cubic (FCC) structure. The biosynthesized Ag-NPs were evaluated for their antimicrobial efficiency using the agar well diffusion method. The antibacterial activity of Ag-NPs was more potent against Gram-negative bacteria than Gram-positive bacteria. Ag-NPs synthesized from Lactobacillus plantarum recorded the maximum activity against Escherichia coli (ATCC® 10536™) and Pseudomonas (ATCC® 27853™) bacteria, while those synthesized from Lactobacillus brevis recorded the maximum activity against Escherichia coli (ATCC® 35218™). Ag-NPs synthesized from Lactobacillus plantarum and Lactobacillus brevis showed antifungal activity against Candida albicans (ATCC® 10231™). The effect of these nanoparticles on Escherichia coli (ATCC® 10536™) was examined and imaged by a transmission electron microscope that indicated damage to the plasma membrane and cell wall. In conclusion, the biosynthesized Ag-NPs have applications as antimicrobial agents in the medicine and food industry.