Objectives: This study was held to evaluate the antibacterial activity of Silver nanoparticle when used as surface treatment on experimental infected dentine surface with Streptococcus mutant and lactobacillus acidophilus biofilm model. Materials and methods: Two different type of silver nanoparticle where prepared with two different nanoparticle size first preparation small and large size of Silver nanoparticle in ethanolic solution (ES and EL groups) and second preparation small and large size of Silver nanoparticle in 2% chlorhexidin (CS and CL groups). Three different methods were used for characterization of Silver nanoparticles. The minimum inhibitory concentrations (MIC) against planktonic bacterial cell were performed using the agar diffusion method. The microbial growth profile was determined by the liquid microdilution method using transparent 96-well microplate. The minimum bactericidal concentration (MBC) of Silver nanoparticle which able to reduced colony forming unit CFU counts as well as ability to inhibits metabolic activity using MTT assays, and lactic acid production using high performance liquid chromatography HPLC and detection change that occur within bacteria using transmission scanning electron microscope TEM. Qualitatively and quantitatively analysis of Resin-dentin interfaces were performed using a Scanning Electron Microscope (SEM) and Energy Dispersive Analytical X-ray (EDAX). Results: Highly monodispersed uniformly distributed spherical Silver nanoparticles for both ES and EL groups while and pseudospherical nanoparticle with aggregation in both CS and CL groups. The minimum inhibitory concentrations (MIC) against S. mutans and L. acidophilus planktonic cell were For CS groups show largest inhibition zone followed by ES groups then EL groups and finally CL groups. Minimum bactericidal concentrations (MBC) against S.mutans and L.acidophilus biofilm show that For CS groups have more potent antibacterial action followed by ES groups opposite to EL and CL show colonies in all agar plate at all different concentration with lower antibacterial action. Silver nanoparticle show total inhibition of metabolic activity and lactic acid production as well as formation of pit and destruction of cell. Ultramorphological examination of resin-dentin interface showed thick hybrid and adhesive layer with short and long resin tag and X -rays microanalysis EDAX show that silver nanoparticle flow within adhesive layer and into dentinal tubule. Conclusion: 1. Synthesization of spherical nanoparticles with microwave irradiation is a rapid and promising technique. 2. Characterization of the synthesized material by features like Size, shape, and peak of absorption greatly influences the experiment conditions. 3. The antibacterial activity of the nanoparticles is concentration and size dependent. 4. The antibacterial potency significantly increased with higher silver nanoparticle concentration specially when dealing with dentin biofilm model.