Objectives This article highlights the biological synthesis of silver nanoparticles (AgNPs) with their characteristic analysis, and it focuses on the application of synthesized NPs against multidrug resistance (MDR) bacteria. A cytotoxicity study was performed to assess the biocompatibility. Methods Silver nanoparticle (AgNPs) formation was confirmed by different characterization methods such as UV–Vis spectrophotometer, Dynamic light scattering (DLS)- Zeta, Fourier transform infrared (FTIR), and Transmission electron microscope (TEM). The antimicrobial activity of the AgNPs was checked against various bacterial strains of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), and Klebsiella pneumonia (K. pneumonia) by disc diffusion, minimum inhibition concentration test (MIC), and kinetic studies. The cytotoxicity of NPs against the Vero cell line was studied by cytotoxic assay. Results The primary analysis of the formation of nanoparticles (NPs) was made by UV–Vis spectrophotometric analysis at 400 nm. At the same time, the efficient capping checked by FTIR shows the presence of a functional group at different wavelengths 3284, 1641,1573,1388,1288, and 1068 cm−1. At the same time, the transmission electron microscopic analysis (TEM) and DLS show that the shape and size of the synthesized NPs possess an average size of around ∼10–30 nm with spherical morphology. Further, the zeta potential confirmed the stability of the NPs. While the yield of NPs formation from silver salt was determined by an online yield calculator with the EDX analysis results. Synthesized NPs showed bactericidal effects against all the selected MDR pathogens with nontoxic effects against mammalian cells. Conclusion Our findings indicate the remarkable antimicrobial activity of the biologically synthesized AgNPs, which can be an antimicrobial agent against multi-drug-resistant bacteria.