This work targets evaluating antimicrobial activity of two types of silver nanoparticles which were prepared within network of two hydrogels composed of PVP/Acrylamide/Acetic acid/Glycerol (AgNP1) and PVP/Acrylamide/Acetic acid (AgNP2) via gamma radiation dose at 25 kGy. Furthermore, HRTEM, was used to investigate the prepared AgNPs with average size around 17.5 and 22.5 nm for AgNP1 and AgNP2 respectively. Silver nanoparticles antibacterial and genotoxic effects were examined on (Escherichia coli ATCC 8739, Salmonella enterica ATCC 14028 and Shigella sonnei ATCC 29930) as examples for gram negative bacteria and one gram-positive bacteria (Staphylococcus aureus ATCC 6538) as tested organisms.Silver nanoparticles (AgNP1 and AgNP2) showed a strong antibacterial activity against the growth of the four bacteria used. Gram-negative bacteria are more affected than gram-positive bacteria. and the most sensitive is Shigella sonnei and AgNP1 is more effective than AgNP2. Effect of AgNP1 and AgNP2 on the DNA of the four bacteria was examined using 11 ISSR primers which produced 35 and 29 polymorphic bands (disappeared and new band appeared) in E. coli, 18 and 17 polymorphic bands in Salmonella enterica and 34 and 32 in Shigella sonnei, respectively. In contrast, gram-positive bacteria Staphylococcus aureus displayed a largest polymorphic bands numbers (38 and 49 bands) comparing to gram-negative. Additionally, all samples treated with AgNP1 or AgNP2 showed a decrease in Band Sharing Index (BSI) and Genetic Template Stability (GTS) according to ISSR profile analysis.