Abstract: Background and Objectives: The use of bio-nanomaterial as a result of environmental remediation procedure, reliability and eco-friendliness has recorded an outstanding development in nanotechnology science. This work studied the biosynthesis of silver nanoparticles (AgNPs) by Lactococcus lactis TNM-B1from fermented tigernut milk and its antibacterial properties against clinical isolates. Materials and Methods: The AgNPs were characterized by visual observation such as; UV-visible spectroscopy (absorption peak between 400-450 nm for the cell free extract and silver nitrate ratios (1:1, 1:4, 1:9, 3:7) at different concentrations of metal ions (1, 2 and 3 mM) and control, without the metal ions), Fourier Transform Infra-Red Spectroscopy (FTIR) and X-ray Diffraction (XRD). Results: The AgNPs characterized by visual observation revealed the ability of the microbial system to form extracellularly silver nanoparticles at 35°C, 160 rpm and 72 h. The UV-visible spectroscopy showed peak at 400 nm which confirmed the presence of nanoparticles of cell-free extract and silver nitrate. The FTIR spectroscopy ascertained the presence of protein functional group as a stabilizing agent and XDR spectra revealed several peaks over the spectrum of 2θ values of 32.1, 46.2, 57.6 and 78.0° corresponding to the 111, 200, 220 and 311 planes as nanocrystals, respectively and AgNPs structural elucidation from high-resolution scanning electron microscopy appeared as convex light core, mono-dispersed with average 15.26 mm width diameter. Conclusion: This study provided an insight into the potential use of nanoparticles as an alternative choice drug against antibiotic-resistant strains. It showed significant antimicrobial activity against Gram-negative (Klebsiella pneumonia, Salmonella typhi, Escherichia coli and Pseudomonas aeroginosa) except Proteus mirabilis compared with ciprofloxacin having observable zones of inhibition.