The plant-derived chemicals present in the leaf extract of Gymnema sylvestre (LEGS) commonly known as "Gurmer" has been utilized for the synthesis of Gymnema sylvestre conjugated silver nanoparticles (GS-AgNPs) at room temperature without any auxiliary reducing and capping agents. Spherical shaped GS-AgNPs of 26.6 nm average diameters were observed. A selective dose dependent antimicrobial activity of the freshly synthesized GS-AgNPs on both gram-positive Staphylococus aureus and gramnegative Proteus vulgaris strain showed the same minimum inhibitory concentration (MIC) value at 50 μg/mL, as revealed by in-vitro MIC assay. Zone inhibition study carried out by Disk agar diffusion (DAD) test showed that the GS-AgNPs is sensitive on both the bacteria cell having a comparatively better antimicrobial activity towards Gram-negative over Gram-positive bacteria. The synthesized GS-AgNPs showed very low toxic effect in normal human cells. Dichlorodihydrofluorescein diacetate (DCDHFDA) and ethidium bromide-acridine orange (EB-AO) staining studies revealed that the GS-AgNPs mainly produces Reactive Oxygen Species (ROS) by altering its electron transport chain (ETC) system and it leads to cell death or necrosis through apoptosis of bacterial cells.