The increasing demand for nutraceuticals indicates that consumers are more concerned about a particular diet associated with good health and lower risk for certain ailments. Mushrooms can be used as functional food and as a source of nutraceuticals. The present study on the nutritional and biochemical characterization of wild and commercial mushrooms collected from Pakistan. Wild Ganoderma lucidum was collected from Jinnah gardenFaisalabad, while two commercial locally cultivated mushrooms Pleurotus ostreatus and Volvariela volvacea and two commercially available exotic mushrooms Lentinus edodes and Hericium erinaceus were collected from local market. Proximate analysis of the selected mushrooms showed that protein, carbohydrate and fiber contents were present in significant amounts whereas the values were low for fats and energy. Crude protein contents were higher in V. volvacea and fiber contents were much higher in G. lucidum. Bioaccumulation capabilities studies of mushrooms revealed that the selected mushrooms are good bioaccumulators of macro and micronutrients; making them good source of essential and non-essential minerals. Phosphorous, potassium, magnesium and Iron were prevalent in V. volvacea whereas calcium and sodium were significantly higher in P. ostreatus. Wild G. lucidum contained more potassium and low sodium. Amino acid profile showed eight essential and nine non-essential amino acids were present in all the selected mushrooms. Glutamine, aspratate, arginine and tyrosine were found in significant amount in P. ostreatus whereas V. volvacea contained proline, cystine and alanine in high concentration. For the assessment of antioxidant, antimicrobial, cytotoxic (brine shrimp) studies the mushrooms were extracted in ethanol and further fractionated in different solvents with ascending polarity (n-hexane, dichloromethane, ethyl acetate and water). The water fractions exhibited good antioxidant and antimicrobial potential. V. volvacea and H. erinaceus had high phenolic contents while flavonoid contents were observed higher in P. ostreatus. Selected mushrooms were found nontoxic against Brine shrimps nauplii (Artemiasalina). Mushrooms extracts and fractions showed activity indicative of thrombolytic and anticancer properties, which were directly proportional to concentration, time of incubation and amount of extract. Mushrooms showed inhibition against tyrosinase and α-glucosidase enzymes. P. ostreatus was the best tyrosinase inhibitor with least IC50 value among the selected commercial mushrooms whereas α-glucosidase activity was very high in G. lucidum. Phyto-constituents (phenolic acids, tocopherol and lutein) were also identified and quantified by HPLC. Gallic acid, chlorogenic acid, p.cumaric acid, caffeic acid and ferulic acids were more frequent in H. erinaceus and V. volvacea. Fatty acids profile by GC/MS recorded that unsaturated fatty acids were more prevalent over the saturated fatty acids except G. lucidum, which contained more saturated fatty acids. Monosaccharide linkage analysis showed that glucose is the main sugar, while small amounts of D-galactose and D-mannose were also present. Methylation analysis revealed the presence of (1,3;1,4)-β-glucan as major components and xyloglucan and glucosamannan as minor components. The results from our studies confirm similar reports by others as well as showed indicator compounds that could have been responsible for their activity against infectious diseases caused by microbes, acclaimed traditional system of medicine. Further studies to substantiate our findings and their development into healthy nutritious food are recommended.