In this research, the synthesis of stable silver nanoparticles by the standard antibiotic reduction method was investigated. The Nalidixic acid act as the reducing and stabilizing agent for the synthesis of silver nanoparticles. The aqueous extract of standard antibiotic Nalidixic acid was mixed with 1mM of Silver nitrate to reduce the metal salt to metal nanoparticles. Synthesized silver nanoparticles (AgNPs) particles were confirmed by analysing the excitation of surface plasmon resonance (SPR) using UV–vis spectrophotometer at 420 nm. The average sizes of the silver nanoparticles were 80 nm to 90 nm as determined through scanning electron microscopy (SEM). Energy-dispersive X-ray spectra (XRD) of silver nanoparticles revealed that the silver was in pure form. These synthesized nanoparticles were found to be highly toxic against human pathogens. All the microbial strains show higher sensitivity to the higher concentration (30 μL) for the test sample when compared to the positive control except B3, B4 and B6. The higher (30 μL/disc) concentration of sample got greater sensitivity than (15 μL/disc) lower concentration in all the tested microorganisms.