The use of antimicrobial drugs has decreased the frequency of infectious ailments. But their widespread use has generated drug-resistant microbes, so there is need to develop new rapid acting antibiotics with less side effects. The silver nanoparticles have been recognized as efficient broad spectrum antimicrobial agents. It uses mushroom glucans as reducing and capping agent to decline its toxicity. The present study was primarily, on the nutritional, biochemical and nutraceutical characterization of mushroom and ultimately, on the green synthesis of silver nano-particles with mushroom glucans and then characterization, antimicrobial and biochemical analysis through in vitro and in vivo studies. The four different Pleurotus spps. including P1: Pleurotus ostreatus, P3: Pleurotus sajor-caju, P6: Pleurotus sapidus and P8: Pleurotus columbinus were collected from mushroom culture bank of Medicinal Mushroom Laboratory, Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan. Proximate analysis of the selected mushrooms depicted the protein (19.07-30.15%), ash (2.1-9.14%), carbohydrate (65.66-82.47%) and fiber contents (38.5-46.3%) were present in significant amounts whereas fats (1.04-6.01%) were in low concentration. All the mushrooms showed potential total phenolic contents (TPC), total flavonoid contents (TFC), DPPH radical scavenging and reducing properties. Selected mushrooms showed DNA protection against H2O2, by use of calf thymus (ctDNA). A moderate antimicrobial potential exhibited by all mushrooms against selected bacterial (E. coli, P. multocida, B. subtilis and S. aureus) and fungal species (A. niger, A. flavus, F. solani and H. maydis). Fatty acids profile by Gas chrometography-Mass spectroscopy (GC-MS) showed that the unsaturated fatty acids were more prevalent over the saturated fatty acids in targeted Pleurotus spps. Quantitative protein analysis depicted h i g h concentration of proteins (33.47-45.78 mg/g) in selected Pleurotus spps. High performance liquid chromatography (HPLC) analysis expressed variable concentration of sugars and organic acids present in the Pleurotus spps. Quantitative ascorbic acid analysis depicted that they were significantly higher (4.90-6.97 mg/g) in chosen Pleurotus spps. High performance liquid chromatography (HPLC) analysis of α-tocopherols and β-carotenes showed that they were present in targeted Pleurotus spps. Macro and micronutrients studies showed that all four Pleurotus spps. have minerals [Na (19.09-39.2 mg/100g) and K (4.84-11.98 mg/100g)] micronutrients [Ca (1.33-11 mg/100g), Mg (1.4-2.1 mg/100g) and P (2.1-4.3 mg/100g)] and xxv trace elements [Cu (10.2-15.42 mg/100g), Mn (2.1-7.4 mg/100g), Zn (37.5-54.6 mg/100g) and Fe (12.1-15.2 mg/100g)] while all were deficient of heavy metals like Pb. Green synthesis of silver nano-particles performed by glucans from selected Pleurotus spps. Extracted glucan and synthesized Glucan-AgNPs confirmed by Ultraviolet-Visible (UV-Vis) spectra. Size of Glucan-AgNPs determined by Dynamic light scattering. The Glucans characterized by Fourier transformation infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM) and X-ray diffraction (XRD) and Glucan-AgNPs were characterized by FT-IR, Scanning electron microscopy-Energy dispersive spectroscopy (SEM-EDS), Transmission electron microscopy (TEM) and XRD. In vitro studies showed that Glucan- AgNPs revealed excellent antimicrobial, biofilm inhibition and anti-cytotoxic potentials. In vivo studies performed on adult albino rats. The glucan-AgNPs significantly restored the physical (Temperature), hematological (Total leukocyte count, neutrophil (%age) count and ESR) and biochemical (C-reactive proteins) parameters elevated after induction of infection. Histological examination revealed that there was no deposition of silver in small intestine, skin, liver and kidney.