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In the present study, silver nanoparticles (AgNPs) were synthesized using both the chemical and biological methods and conjugated with <i>Pyrenacantha grandiflora</i> extracts. These were then characterized and evaluated for antimicrobial activities against multi-drug resistant pathogens, such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), <i>Klebsiella pneumonia,</i> and <i>Escherichia coli</i>. Nanoparticles were analyzed with UV-visible spectrophotometer, transmission electron microscopy (TEM), and energy dispersive X-ray analysis (EDX). Silver nanoparticles, <i>P. grandiflora</i> extracts, and the conjugates were also analyzed with Fourier transform infrared spectroscopy (FTIR). As a result, quasi-sphere-shaped AgNPs with sizes ranging from 5 to 33 nm and spherically shaped AgNPs with sizes ranging from 3 to 25 nm were formed from chemical and biological synthesis, respectively. A well diffusion assay showed that the activity of silver nanoparticles was most improved with acetone extract against all tested bacteria with diameters in the range of 19–24 mm. The lowest MIC value of 0.0063 mg/mL against MRSA was observed when biologically synthesized AgNPs were conjugated with acetone and water extracts. Chemically synthesized silver nanoparticles showed the lowest MIC value of 0.0063 mg/mL against <i>E. coli</i> when conjugated with acetone and methanol extracts. This study indicates that silver nanoparticles conjugated with <i>P. gandiflora</i> tubers extracts exhibit strong antibacterial activities against multi-drug resistant bacterial pathogens. Therefore, biosynthesized conjugates could be utilized as antimicrobial agents for effective disease management due to the synergistic antibacterial activity that was observed.